Water-soluble aie luminogens for monitoring and retardation of fibrillation of amyloid proteins

ABSTRACT

Compounds that exhibit aggregation induced emission (AIE), and more particularly to water-soluble conjugated polyene compounds that exhibit aggregation induced emission. The conjugated polyene compounds can be used as bioprobes for DNA detection, G-quadruplex identification, and potassium-ion sensing. The polyenes also can be utilized as an external fluorescent marker to study conformational structures, to monitor folding processes of label-free oligonucleotides with G-rich strand sequences, and to visualize DNA bands in PAGE assay. The polyenes have applications in high-throughput anticancer drug screening and are useful for the development of efficient anti-cancer drugs. Furthermore, the present subject matter can also be used to monitor fibrillation of amyloid proteins and to facilitate the storage and delivery thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation-in-Part application of U.S. patentapplication Ser. No. 13/359,514, which was filed on Jan. 27, 2012 andwhich claims the benefit of Provisional Application No. 61/457,207 filedon Jan. 31, 2011, and is also a Continuation-in-Part application of U.S.patent application Ser. No. 12/453,892, which was filed on May 26, 2009,which claims the benefit of Provisional Application No. 60/873,431 filedon Dec. 8, 2006, and to Provisional Application No. 60/929,364 filed onJun. 25, 2007, and to Provisional Application No. 61/071,928 filed onMay 27, 2008, and which is also a Continuation-in-Part application ofU.S. patent application Ser. No. 12/000,130, which was filed on Dec. 10,2007, now U.S. Pat. No. 7,939,613, and which is also aContinuation-in-Part application of U.S. patent application Ser. No.11/408,846, which was filed on Apr. 21, 2006 and claims priority to U.S.Provisional Application No. 60/673,562 filed on Apr. 22, 2005. All ofthese applications are hereby incorporated by reference in theirentirety.

TECHNICAL FIELD

The presently described subject matter relates generally to compoundsthat exhibit aggregation induced emission (AIE), and more particularlyto water-soluble conjugated polyene compounds that exhibit aggregationinduced emission. The conjugated polyene compounds can be used asbioprobes for DNA detection, G-quadruplex identification, andpotassium-ion sensing. The polyenes also can be utilized as an externalfluorescent marker to study conformational structures, to monitorfolding processes of label-free oligonucleotides with G-rich strandsequences, and to visualize DNA bands in PAGE assay. The polyenes haveapplications in high-throughput anticancer drug screening and are usefulfor the development of efficient anti-cancer drugs. Furthermore, thepresent subject matter can also be used to monitor fibrillation ofamyloid proteins and to facilitate the storage and delivery thereof

BACKGROUND

Fluorescence (FL) techniques have emerged as a mainstream research anddevelopment area in science and engineering, particularly in the fieldof biochemical and biological science. Currently, fluorescent moleculesare used as probes for DNA sequencing, fluorescence-activated cellsorting, high-throughput screening, and clinical diagnostics.

Fluorescence-based techniques offer high sensitivity, low backgroundnoise and broad dynamic ranges. A great number of fluorescent probeshave been investigated and are already widely used in biotechnology.Many of them show favorable spectral properties of visible absorptionand emission wavelength, high extinction coefficients, and reasonablequantum yields. Upon complexation with proteins and DNA, thefluorescence of the bioprobes can be enhanced/quenched and/orred/blue-shifted, thus enabling visual observation of thebiomacromolecular species. Among these, the most useful probes are thosethat act as “turn-on” sensors, whose fluorescence is activated by theanalytes.

Several probes for DNA detection based on fluorescent enhancement havebeen developed such as phenanthridine and acridine derivatives.Middendorf et al. have reported on ethidium bromide (EB), a well-knownphenanthridine derivative, which has already been widely used for DNAsequencing (See, for example, U.S. Pat. No. 4,729,947, U.S. Pat. No.5,346,603, U.S. Pat. No. 6,143,151, and U.S. Pat. No. 6,143,153). FLenhancement induced by proteins can be attributed to the interactionwith hydrophobic regions of proteins, such as NanoOrange (MolecularProbes, Inc., U.S. Pat. No. 6,818,642) and Nile red (U.S. Pat. No.6,897,297, U.S. Pat. No. 6,465,208), or reaction with amine groups ofproteins in the presence of cyanide or thiols, such as fluorescamine(U.S. Pat. No. 4,203,967) and o-phthaldialdehyde (U.S. Pat. No.6,969,615, U.S. Pat. No. 6,607,918). The FL of cyanine dyes has beenfound to increase dramatically upon complexation with DNA and proteins.(U.S. Pat. No. 5,627,027, U.S. Pat. No. 5,410,030). Haugland et al. havereported unsymmetrical cyanine dyes, which possess superior fluorescentcharacteristics when complexed with nucleic acids (U.S. Pat. No.5,436,134). The SYPRO® dyes are merocyanine dyes that are essentiallynon-fluorescent when free in solution but become intensely fluorescentin hydrophobic environments (e.g. SYPRO® Red and SYPRO® Orange dyes ofMolecular Probes, Inc., U.S. Pat. No. 6,914,250, U.S. Pat. No.6,316,267). Water-soluble cyanine dyes, such as Cy3 and Cy5, arecommonly used in labeling of DNA or RNA for microarray (Y. R. Iyer etal., Science, 1999, 283, 83). Cy3 and Cy5 have merits of highfluorescence intensity and emission even in the solid state; however,they are quite unstable and show insufficient detection sensitivity(U.S. Pat. No. 7,015,002).

As described in U.S. Pat. No. 7,109,314, a good fluorescent dye shouldpossess a high fluorescent quantum yield and molecular absorptioncoefficient, as well as good solubility in aqueous media and stabilityunder ambient conditions. However, most of the dyes discussed above arelipophilic, which are, at best, only dispersible in aqueous media. Forexample, Nile Red, a dye used to stain proteins, should be firstdissolved in acetone and then mixed rapidly with water immediately priorto use (J. R. Daban et al., Anal. Biochem, 1991, 199, 169).

Additionally, substantially all of the above-described fluorescent dyessuffer from the problem of aggregation-caused quenching (ACQ). Due totheir lipophilic character, these fluorescent dyes are prone toaggregate when dispersed in aqueous media or when bound to biologicalmacromolecules. The close proximity of the chromophores often induces anon-radiative energy transfer mechanism that results in self-quenchingof the luminescence. This self-quenching drastically reduces the dyes'fluorescent signal thereby prohibiting their use as efficient bioprobesor biosensors.

Substantial effort has been made to mitigate aggregate formation ofthese dyes (J. R. Lakowicz, et al. Anal Biochem, 2003, 320, 13).However, only a small number of researchers have focused on the designand synthesis of novel organic molecules or polymers that do not sufferfrom fluorescent quenching, and moreover, even display enhanced lightemission upon aggregation.

Recently, some non-emissive dyes have been induced to emit efficientlyby aggregate formation, the exact opposite of ACQ. AIE molecules with ahigh quantum yield Φ_(F) (up to 0.85) and various emission colors (blue,green, yellow and red) have been reported. While the AIE dyes have beenused for the construction of efficient optical and photonic devices, thepossibility of employing them as bioprobes for detecting biopolymers hasbeen virtually unexplored. Accordingly, there remains a great need forwater-soluble “light-up” compounds and probes, for example, for thedetection of biomacromolecules such as DNA and proteins.

Many known fluorescent materials accomplish the detection of saccharidesby the competing intramolecular interaction of an amine functionalitywith a boronic acid pendant. Less effort has been spent on the detectionof other biological compounds. Furthermore, vapor-sensing compounds anddevices are often manufactured from expensive platinum salts andcomplexes and/or in combination with palladium. They are based mainly ona color shift from dark-red to light-red, making it difficult tovisually sense the color shift. Sensors exhibiting an on-off change intheir luminescent color rather than a color shift will be thus not onlyadvantageous but also more sensitive. To date, the only known “on-off”example was shown by Kato (U.S. Pat. No. 6,822,096), who utilized theluminescence change from the invisible near-infrared to the visible redof binuclear platinum (II) complexes. However, these complexes onlyshift the emitted wavelength out of the visible spectrum.

Fluorescent materials, including inorganic semiconductor quantum dots,organic and metallorganic dyes, dye-doped silica or polymer particles,have currently attracted great attention in a wide variety ofhigh-technology applications such as high-throughput screening,ultra-sensitive assays, optoelectronics, and living cell imaging.Colloidal quantum dots (hundreds to thousands of atoms) aretraditionally made from crystals of IIA-VIA or IIIB-VB elements (PbS,CdSe, etc.) or other semiconductors. The heavy metals therein areintrinsically toxic to the researchers and the experimental systems(e.g., living cells), as well as generating a toxic waste stream intothe environment. Organic and metallorganic dyes generally consist ofπ-conjugated ring structures such as xanthenes, pyrenes or cyanines,with emissions across the spectrum from UV to the near infrared(˜300-900 nm) and may be fine tuned to particular wavelengths orapplications by changing the chemistry of their substituent groups. Thesize of individual dye molecules is very small (˜1 nm), which causesnon-specific labeling and high background signals as dyes diffuse awayfrom their intended targets. Spectrally, organic dyes tend to havefairly wide absorption and emission spectra (FWHM˜50 nm), which can leadto spectral overlap and re-absorption when using multiple dye speciessimultaneously. In normal use, dye molecules are exposed to a variety ofharsh environments and often suffer from photobleaching and quenchingdue to the interactions with solvent molecules and reactive species suchas oxygen or ions dissolved in solution.

In order to create more robust emitters with enhanced brightness andstability, composite nano- and micro-particles consisting of dyemolecules and silica or polymer matrix have been developed. Thus theencapsulated dye molecules can be protected from external perturbations,with reduced stochastic blinking, photobleaching, and quenching.Dye-loaded polymer particles are superior to their silica counterpartsin terms of the versatile chemical compositions, tunable surfacechemistry suited for biocompatibility and bioconjugation, facilepreparation, and easy control of the particle size and sizedistribution.

Gao et al. have incorporated pyrene dyes into polystyrene particlesusing a normal microemulsion approach, leading to a 40-fold increase inemission intensity with respect to the pure dye at the identicalconcentration (H. Gao et al., Colloid Polym. Sci. 2002, 280, 653).Dinsmore et al. swelled poly(methyl methacrylate) particles and absorbeda rhodamine dye into them for usage in a confocal microscopic study ofcolloidal dispersions (A. D. Dinsmore et al., Appl. Opt. 2001, 40,4152). U.S. Pat. No. 5,716,855 discloses fluorescent particlescontaining anthracene- or naphthacene-derived dyes aiming to theapplication as biological markers.

Up to now, most of the organic dyes commercially available, includingthe above mentioned dyes as well as ethidium, Nile red, fluorescamine,o-phthaldialdehyde, cyanine dyes, etc. are emissive only in theirsolution state, whereas emission is quenched in aggregation states(e.g., high dye concentration state, film state, solid state, etc.).This is attributed to the mechanism of non-radiative energy transferbetween the closely packed chromophores, thus resulting inself-quenching of the fluorescence. Thus, the loading concentration ofdyes in the polymer particles cannot be sufficiently high andaccordingly the intensity of fluorescence is considerably limited.

With respect to polymers for dye encapsulation, the currently availablespecies are mainly hydrophobic polystyrene and less hydrophobicpoly(methyl methacrylate), as mentioned hereinabove. The hydrophobicnature of these particles commonly leads to clustering and non-specificbinding of biological materials, which considerably limits theirapplication in the aqueous environment of biology and other fields.Additionally, these particles are prepared and dispersed in an organicsolvent. For example, Hu et al. prepared poly(methyl methacrylate)fluorescent particles through dispersion polymerization in a mixture ofhexane and ethanol (H. Hu et al., Langmuir 2004, 20, 7436). Thesolvent-dispersible polymer particles are difficult to disperse stablyin aqueous media.

Recent studies on biomacromolecules aid the understanding ofpathogenesis of numerous diseases as well as development of effectivetherapeutic agents. For example, fibrillation of amyloid proteins isrecognized as a pathological hallmark of many neurodegenerativediseases, including Alzheimer's disease, Parkinson's disease, Priondisease, and Huntington's disease. Insulin is a well-established modelof amyloid fibrillation and its amyloid fibrils are found at frequentinjection sites of diabetic patients and have been suggested asindicative of Parkinson's disease. Therefore, monitoring insulinaggregation and/or other amyloid proteins facilitates the understandingof pathogenesis of many neurodegenerative diseases or other diseasesassociated therewith, thereby developing effective diagnostic tools andtherapeutic agents.

Amyloid fibril formation of insulin has been studied by a variety ofspectroscopy and microscopy techniques including transmission electronmicroscopy (TEM), atomic force microscopy (APM), real-time lightscattering, stopped-flow turbidimetry, X-ray diffraction, fluorescence,circular dichroism (CD), and NMR spectroscopy. Among them, fluorescencetechnique is the most commonly used method on intrinsic fluorescence ofproteins. For example, Thioflavin T (ThT) is a standard fluorescenceprobe for amyloid assay. Despite its widespread use, it suffers from anumber of drawbacks, such as small stokes shift, low specificity, poorsensitivity, false-positive response, poor reliability, incapability ofcatching oligomeric intermediates, and unsuitability for kinetic study.Many of the other fluorophores for amyloid detection also containelectron donors and acceptors, between which intramolecular chargetransfer occurs. Such fluorophores are sensitive to the hydrophobicityof the environment and their emissions are intensified upon binding tohydrophobic regions of amyloids rich in β-sheet structure. However, whenmultiple fluorophore molecules are accumulated in a hydrophobic patch ofprotein, π-π interaction between their stacked aromatic rings occurs,which promotes the formation of such detrimental species as excimers andexciplexes. This can lead to severe emission self-quenching, making thefluorophores unsuitable for quantitative analysis.

In addition to the problems in monitoring, insulin fibril formation hasbeen a nuisance in delivery and long-term storage for treatment ofdiabetes because insulin can form amyloid fibrils in vitro under certaindestabilizing conditions, such as elevated temperature, low pH,increased ionic strength and exposure to hydrophobic surfaces. It isalso generally believed that dissociation of the native associatedstates of insulin (i.e., dimers, tetramers and hexamers) into monomersis a prerequisite for fibril formulation. The monomers undergo partialunfolding into intermediate states, in which they re-associate intostable and fibrous amyloid aggregates. These destabilizing conditionslead to an early maturity of amyloid fibrils, which does not favor thelong-term storage and delivery of insulin. Therefore, inhibitors forprotein aggregation are also of great significance in developingeffective therapeutic agents for diabetes or for diseases treatable bysimilar biomolecules.

Functional kidneys are capable of removing wastes from the body,regulating electrolyte balance and blood pressure, and stimulating redblood cell production. Kidney diseases are a major cause of healthproblems world-wide. E.g. >20 million Americans-1 of 9-adults havechronic kidney disease (CKD). Another 20 million more Americans are atincreased risk (US National Kidney Foundation). Each year in the UnitedStates, more than 100,000 people are diagnosed with kidney failure(ESRD: End-Stage Renal Disease). The high-risk groups for kidney diseaseinclude diabetes and hypertension patient. Most kidney diseases do notcause noticeable symptoms until very late. Nearly 50% of people with anadvanced form of kidney disease even don't know. However, certainchanges in the urine can be seen earlier, which may suggest problemswith kidneys or urinary tract. There are over a hundred different typesof proteins in the blood and the kidneys are very good at keeping themfrom entering the urine. Most of the proteins that make it into theurine are reabsorbed, chewed up and returned to the blood. As a result,less than 150 mg (30 mg/L) of protein is normal lost in the urine perday. A higher level of protein loss in the urine is called proteinuriaand may mean there is a kidney disease. Determination of urinary proteinis of major clinical importance because it readily reflects kidneyfunctionality.

Accordingly, there is a need in the art for new sensors useful fordetecting/sensing a wide variety of biomacromolecules. Sensors based ondetecting fluorescence of an analyte such as a biomacromolecule arehighly sensitive, thereby lowering detection limits. Sensors that havethe capability to quantitatively analyze kinetics of biomacromoleculesare desired. Notably, fluorescent markers that enable the monitoring ofamyloid fibrillation and compounds that inhibit fibrillation are mostdesired.

SUMMARY

The presently described subject matter is directed to water-solubleconjugated polyenes which exhibit aggregation induced emission (AIE) andare useful as bioprobes and for manufacturing sensors. The emissioncolor of these water-soluble conjugated polyenes ranges from blue to redarising from the different chromophoric structures. They exhibit AIE(i.e., increased fluorescence) upon addition of a non-aqueous solvent.Their luminescent behavior features the AIE phenomenon, which turns thedyes (water-soluble conjugated polyenes) from faint-emitters whenmolecularly dissolved in an aqueous solvent, i.e., water, into strongluminophors when aggregated or in the solid state. Stated differently,when the compounds are dissolved in aqueous solvents, they aresubstantially non-emissive (“off”) while when a non-aqueous solvent isadded, they aggregate and emit intensely (“on”). The quantum efficiencyincreases when the amount of non-aqueous solvent is increased. Thepresently described water-soluble conjugated polyene compounds areuseful as “turn-on” fluorescence sensors. In addition, the presentlydescribed subject matter is directed to water-dispersible fluorescentpolymer particles, i.e., micro-particles and/or nanoparticles,comprising the described water-soluble conjugated polyenes, for example,a tetraphenylethylene (“TPE”).

In an embodiment, the present subject matter relates to a water-solubleconjugated polyene compound comprising a backbone structure of a formulaselected from:

whereinR and R′ are independently selected from H, X, B(OH)₂, (X)_(n)COOR″,(X)_(n)COOH, (X)_(n)NH₂, (X)_(n)NHR″, (X)_(n)NR″₂, (X)_(n)N⁺R″₃Br⁻,(X)_(n)OH, (X)_(n)SH, (X)_(n)SO₃ ⁻Na⁺, (CH₂)_(n)P⁺R″₃Br⁻,

X is selected from (CH₂)_(n), O(CH₂)_(n), NH(CH₂)_(n), N[(CH₂)_(n)]₂,(CH═CH)_(n) and (OCH₂CH₂)_(n); and R″ is selected from R, R′,(CH₂)_(n)CH₃, CONH—X—, COO—X—, C₆H₅—R, —CH₂—C₆H₅, and C₆H₅; n=0 to 20,and salts thereof, and wherein the compound is water-soluble andexhibits aggregation induced emission.

In an embodiment, the present subject matter relates to a water-solubleconjugated polyene compound, wherein the compound does not exhibitaggregation induced quenching.

In an additional embodiment, the present subject matter relates to amethod for detecting the presence or absence of a targetbiomacromolecule in a biological sample, comprising contacting thebiological sample with the water-soluble conjugated polyene compound,and detecting luminescence.

In a further embodiment, the present subject matter relates to a methodfor detecting the presence or absence of a target biomacromolecule in abiological sample, wherein the biological sample is selected from thegroup consisting of a tissue sample, a cell sample, blood, saliva,spinal fluid, lymph fluid, vaginal fluid, seminal fluid, and urine.

In an embodiment, the present subject matter relates to a sensor devicefor detecting the presence or absence of a target biomacromolecule,comprising a holder and a detecting molecule comprising thewater-soluble conjugated polyene compound, the detecting molecule beingheld in place by the holder and being accessible to the target moleculeor substance.

In a further embodiment, the present subject matter relates to a sensordevice, wherein the luminance of the detecting molecule increases uponcontact with the target biomacromolecule.

In another embodiment, the present subject matter relates to a sensordevice, wherein the holder is a container and the detecting molecule isdisposed inside the container; the container having one or more openingsor orifices to allow access to the detecting molecule by the targetmolecule.

In yet another embodiment, the present subject matter relates to asensor device, wherein the holder is a surface on which the detectingmolecule is coated in a thin film.

In an embodiment, the present subject matter relates towater-dispersible, fluorescent polymeric particles, comprising orconsisting of a water-soluble conjugated polyene compound of any offormulae I-X; and a polymer comprising one or more ethylenicallyunsaturated monomers.

In an embodiment, the present subject matter relates to a method ofdetecting G-quadruplex formation, DNA, or protein and protein levels ina biological sample comprising contacting the biological sample with thewater-soluble conjugated polyene compound and detecting luminescence.

In another embodiment, the present subject matter relates to a method ofdetecting G-quadruplex formation, DNA, or protein and protein levelswherein the conjugated polyene compound forms a complex with G-richstrand sequences of the biological sample which activates thefluorescence of the polyene.

In a further embodiment, the present subject matter relates to a methodof detecting G-quadruplex formation, DNA, or protein and protein levelswherein a cation is added to the biological sample and polyene mixture.

In another embodiment, the present subject matter relates to a method ofdetecting G-quadruplex formation fluorescence emission intensity ismonitored for any spectral shifts signaling the presence of aG-quadruplex conformation in the folded oligonucleotide.

In a further embodiment, the present subject matter relates to a methodof detecting G-quadruplex formation, DNA, or protein and protein levelsin a biological sample wherein the biological sample is selected fromthe group consisting of a tissue sample, cell sample, blood, saliva,spinal fluid, lymph fluid, vaginal fluid, seminal fluid, and urine.

In another embodiment, the present subject matter relates to a method ofdetecting G-quadruplex formation, DNA, or protein and protein levels ina biological sample wherein the biological sample is urine.

In another embodiment, the present subject matter relates to a method ofdetecting G-quadruplex formation, DNA, or protein and protein levels ina biological sample wherein the protein being detected in the biologicalsample is human serum albumin.

In an embodiment, the present subject matter relates to a method ofdiagnosing a kidney disorder comprising contacting a biological samplewith a water-soluble conjugated polyene compound and detectingluminescence.

In another embodiment, the present subject matter relates to a method ofdiagnosing a kidney disorder wherein the conjugated polyene compoundforms a complex with proteins in the biological sample thereby causingthe conjugated polyene compound to fluoresce wherein the huninescencelevels indicate the levels of protein present in the biological sample.

In yet another embodiment, the present subject matter relates to amethod of diagnosing a kidney disorder wherein the protein beingdetected in the biological sample is human serum albumin.

In a further embodiment, the present subject matter relates to a methodof detecting guanine (G)-rich repeat sequences in a biological samplecomprising running the biological sample through a poly(acrylamide) gelelectrophoresis (PAGE) assay, staining the PAGE assay with awater-soluble conjugated polyene compound, and detecting luminescence.

In an exemplary embodiment, the present subject matter relates to amethod of screening a potential anti-cancer drug for activity includingcontacting said anti-cancer drug with a biological sample having aG-rich DNA sequence that is capable of forming a particular G-quadruplexconformer to form a reaction mixture, adding a water-soluble conjugatedpolyene compound to the reaction mixture of said anti-cancer drug andsaid G-rich DNA, and detecting luminescence. In another embodiment, thewater-soluble conjugated polyene is added to the biological samplebefore the anti-cancer drug is mixed with the biological sample to formthe reaction mixture. The potential anti-cancer drug, biological sampleand water-soluble conjugated polyene can be added in any order and suchwill not affect the determination of the activity of the screenedpotential anti-cancer drug.

In another embodiment, the present subject matter relates to a method ofscreening a potential anti-cancer drug for activity further includingcomparing the detected luminescence from said water-soluble conjugatedpolyene compound in the reaction mixture of said biological samplehaving the G-rich DNA sequence and said anti-cancer rug with aluminescence detected from said water-soluble conjugated polyenecompound in said biological sample having the G-rich DNA sequence alone,wherein said comparing further includes observing a bathochromic shiftas an indicator of the formation of the particular G-quadruplexconformer in the biological sample.

In a further embodiment, the present subject matter relates to a methodof screening a potential anti-cancer drug for activity, furthercomprising comparing the detected luminescence from said water-solubleconjugated polyene compound in the reaction mixture of said biologicalsample having the G-rich DNA sequence and said anti-cancer drug with aluminescence detected from said water-soluble conjugated polyenecompound in a mixture of said biological sample having the G-rich DNAsequence and K⁺ ions, wherein the fluorescence emission profiles betweenthe biological sample with said anti-cancer drug and the biologicalsample with K⁺ ions are similar. The G-quadruplex-inducing ability ofthe screened anti-cancer drug relative to the known G-quadruplexinducer, K⁺ ions can be determined by the fluorescence intensitiesthereof at a specific wavelength, e.g. at 492 nm. In yet anotherembodiment, the water-soluble conjugated polyene compound is1,1,2,2-tetrakis[4-(2-triethylammonioethoxy)phenyl]ethene tetrabromide(TTAPE).

In an embodiment, the present subject matter relates to an anti-cancerpharmaceutical composition comprises a water-soluble conjugated polyenecompound. In another embodiment, said compound is conjugated with aG-quadruplex targeting motif, wherein the G-quadruplex targeting motifis capable of targeting a DNA sequence that is induced by said motif toform a particular G-quadruplex conformer over other G-quadruplexstructures or duplex structures. The G-quadruplex targeting motif may beisolated from an anticancer drug being screened by the present. In afurther embodiment, said compound is a chemically modified TPE. Inanother embodiment, said compound is a chemically modified TTAPE. In yetanother embodiment, one or more of the triethylamine groups of the TTAPEare substituted with other positively charged groups includingpiperidine, pyrazole, piperazine and imidazole so as to obtain achemically modified TTAPE. The chemically modified TTAPE may also act asa lead compound in said pharmaceutical composition because TTAPE itselfhas been shown to have a low efficiency of inducing the formation ofG-quadruplex at certain conditions such as at room temperature.

The presently described series of linear and cyclic π-conjugated organiccompounds (hereinafter polyenes) have been designed and synthesized withdifferent chromophores including tetraphenylethylene, siloles, fulvene,butadienes, and 4H-pyrans. The emission color of these new polyenesranges from blue to red arising from the different chromophoricstructures. Their fluorescent behavior features the AIE phenomenon,which turns the dyes from faint-emitters when molecularly dissolved intostrong luminophors when aggregated or in the solid state. The presentlydescribed AIE-active-molecules are highly specific to amyloid proteinsand exhibit retardation effect on amyloid protein fibrillation. Theexcellent water solubility of the AIE-active-molecules allow backgroundluminescence to be ignored. All these features make the presentlydescribed AIE-active molecules excellent candidates for use as bioprobesfor DNA detection, G-quadruplex identification and potassium-ion sensingas well as in polymeric particles, sensors and detection devices. TheAIE-active-molecules can also be used to study conformational structuresand folding processes, as fluorescent markers to visualize DNA bands inassays and to screen anti-cancer drug as well as for use in anti-cancertherapy. In addition, the presently described AIE-active-molecule's dualcapability to discriminate native and fibrillar forms of amyloidproteins under physiological condition and to retard fibrillation, serveits use as an external tool for quantitative and kinetic analysis ofamyloid fibrillation and as an antiamyloid agent for long-term storageand delivery of amyloid, such as pharmaceutical insulin.

In an embodiment, the present subject matter relates to a method ofmonitoring amyloid protein fibrillation in a biological sample. Themethod includes the use of a water-soluble conjugated polyene compoundto contact with the biological sample and detecting luminescence fromthe compound induced by aggregation when contacting the biologicalsample, and wherein an increase of luminescence indicatesfibrillogencsis. In a preferred embodiment, the conjugated polyenecompound for monitoring amyloid protein fibrillation in a biologicalsample includes a chemical structure of the following formula:

wherein A is selected from a cyanide or any aromatic group; and X is acation. In a preferred embodiment, A is a substituted or unsubstitutedaromatic moiety. In a more preferred embodiment, A is a phenyl. Inanother preferred embodiment, X is selected from the group consisting ofK⁺, Li⁺, Na⁺, Mg²⁺, NH₄ ⁺ and Ca²⁺. In a further preferred embodiment,the conjugated polyene compound for monitoring amyloid protein is sodium1,2-bis[4-(3-sulfonatopropoxyl)phenyl]-1,2-diphenylethene (TPE-SO3)

In another embodiment, the amyloid protein being monitored by the methodof the present subject matter is selected from the group consisting ofinsulin, amyloid beta-peptide, tau, alpha-Synuclein, PrP andpolyglutamine-containing peptides, such as human islet amyloidpolypeptide (hIAPP).

In a further embodiment, the present subject matter relates to a methodof retarding fibrillation of an amyloid protein for long-term storageand the delivery thereof, comprising storing said amyloid protein with awater-soluble conjugated polyene compound. In yet another embodiment,the fibrillation of the amyloid protein being retarded by the method ofthe present subject matter is selected from the group consisting ofinsulin, amyloid beta-peptide, tau, alpha-Synuclein, PrP andpolyglutamine-containing peptides, such as hIAPP. In a preferredembodiment, the amyloid protein is insulin. In a more preferredembodiment, the amyloid protein is pharmaceutical insulin for treatmentof diabetes.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A, 1B, and 1C illustrate absorption and emission spectra of (A)TPE-OMe (10 μM), (B) TPE-OH (10 μM), and (C) TPE-SO3 (5 μM) in pureacetonitrile, pure water, and mixtures of acetonitrile and water. FIG.1A-C shows that the three TPE derivatives are practically non-emissivewhen dissolved but highly emissive when aggregated.

FIG. 2A illustrates the change of fluorescence spectrum of TPE-OHNa₂ (5μM) with addition of BSA in aqueous phosphate buffer (pH=7.0).

FIG. 2B illustrates a plot of fluorescence intensity at 476 nm versusBSA concentration.

FIG. 2C illustrates the linear region of the binding isotherm of TPE-OHto BSA. The FL of TPE-OH is turned on in the presence of BSA (FIG. 2).Its intensity increased with increasing BSA concentration, and in theBSA concentration range of 0-10 μg/ml exhibited a linear relationshipwith a R² value of 0.9974.

FIG. 3A illustrates the change of FL spectrum of TPE-SO3 with additionof BSA in an aqueous phosphate buffer.

FIG. 3B illustrates the plot of FL intensity at 472 nm versus BSAconcentration.

FIG. 3C illustrates the linear region of the (I/I₀-1)-[BSA] plot in FIG.3B. TPE-SO3 shows similar behavior as TPE-OH but better performance. TheFL intensity increase up to 240 times upon binding with BSA. Linearrange in the BSA concentration from 0 to 100 μg/m is given with a R²value of 0.9971.

FIG. 4A illustrates the effect of dye concentration on the FL intensityof buffer solution of TPE-OHNa₂ at 467 nm or TPE-SO3 at 472 nm in theabsence or presence of BSA (10 μg/m).

FIG. 4B illustrates the effect of BSA (100 μg/ml) and/or SDS (1 mg/ml)on the FL spectrum of a buffer solution of TPE-SO3 (5 μM). Conventionalfluorescent dyes suffer from self-quenching at high dye concentrations,whereas the FL of the AIE-active dyes is intensified with increasing dyeconcentration as illustrated in FIG. 4A. The FL of TPE-SO3 solution inthe presence of BSA is diminished by adding surfactants such as sodiumdodecyl sulphate (SDS) in high concentration (1 mg/ml) as illustrated inFIG. 4B.

FIG. 5A illustrates the emission spectra of TPE-C2N⁺ (2.5 μM) in anaqueous phosphate buffer (pH=7) and in the buffers containing 300 μg/mlBSA.

FIG. 5B illustrates a plot of fluorescence intensities of buffersolutions of TPE-C2N⁺ at 462 nm versus concentrations of DNA and BSA.

FIG. 6A illustrates the emission spectra of TPE-C4N⁺ (4) (2.5 μM) in anaqueous phosphate buffer (pH=7) and in the buffers containing 300 μg/mlcalf thymus DNA (“ctDNA”) and 500 μg/ml BSA. FIG. 6B illustrates plotsof fluorescence intensities of buffer solutions of TPE-C4N⁺ (4) at 463nm versus concentrations of ctDNA and BSA. FIGS. 5 and 6 illustrate thatthe FL of cationic TPE derivatives in aqueous solution can is turned onin the presence of BSA or DNA. TPE-C2N⁺ exhibits better affinity toctDNA than BSA while TPE-C4N⁺ gives the opposite result.

FIG. 7A illustrates the emission spectra of TPE-C4N⁺ (2.5 μM) in anaqueous phosphate buffer (pH=7) and in buffers containing 300 μg/mlctDNA and 500 μg/ml BSA.

FIG. 7B illustrates plots of fluorescence intensities of buffersolutions of TPE-C4N⁺ at 463 nm versus concentrations of ctDNA and BSA.The FL of TPE-C4N⁺ and TPE-C2N⁺ is intensified with increased dyeconcentration.

FIG. 8 illustrates increments of fluorescence of TPE-C2N⁺ (8),N+C2-TPE-C2N⁺ (9), TPE-C4N⁺ (10), N+C4-TPE-C4N⁺ (11) when binding with10 μg/ml ctDNA/10 μg/ml RNA diethylaminoethanol (DEAE) salt from torulayeast/10 μg/ml RNA from torula yeast in buffer solution pH=7.Concentration of dyes: 5 μM; excitation wavelength: 350 nm. The fourcationic TPE derivatives display larger FL enhancement in the presenceof DNA than that of RNA. Meanwhile N+C2-TPE-C2N⁺ and N+C4-TPE-C4N⁺ showmuch larger variety of FL than that of TPE-C2N⁺ and TPE-C4N⁺ in thepresence of DNA and RNA.

FIG. 9 illustrates the binding isotherm of N+C2-TPE-C2N⁺ (5 μM) toctDNA/RNA from torula yeast/BSA (plot of the fluorescence intensity at470 nm for ctDNA/RNA, and at 467 nm for BSA) in aqueous phosphate buffer(pH=7.0). FIG. 9 illustrates that N+C2-TPE-C2N⁺ has larger affinity toDNA than to RNA and proteins.

FIG. 10 illustrates the photoluminescence spectra of the water/methanol(6:4) solutions of a PPS—OH (5.7×10⁻⁵ M) in the presence of KOH(8.4×10⁻⁴ M) and BSA. The spectrum of a “pure” BSA solution (0.50 wt %)is shown for comparison. Excitation wavelength: 378 nm. Water-solublesilole derivatives also show this “tum-on” property when binding to BSAin aqueous solutions.

FIG. 11A illustrates the absorption and emission spectra of solutions ofderivative 1 (10 μM) in AN and AN-water mixture (1:99 v/v). The inset isphotographs of solutions of derivative 1 in (a) AN and (b) the AN-watermixture taken under illumination of a UV lamp.

FIG. 11B illustrates the dependence of fluorescence quantum yields ofsolutions of derivatives 1 and 2 on the solvent composition of AN-watermixture λ_(ex)=350 nm.

FIG. 12A illustrates the emission spectra of derivative 4 (2.5 μM) in anaqueous phosphate buffer (pH=7) and in the buffers containing 300 μml⁻¹ctDNA and 500 μml⁻¹ BSA.

FIG. 12B illustrates plots of fluorescence intensities of buffersolutions of derivative 4 at 463 nm vs. concentrations of ctDNA and BSA.

FIG. 13A illustrates the absorption and emission spectra of derivative 4(2.5 μM) in water and a glycerol-water mixture at 25° C.

FIG. 13B illustrates the emission spectra of derivative 4 (2.5 μM)

FIG. 14 illustrates the absorption and photoluminescence spectra ofTPE-COOH in acetonitrile/water mixture (1:99 v/v). TPE-COOHconcentration: 10 μM; excitation wavelength: 346 nm.

FIG. 15 illustrates the light emission of the fluorescent polymerparticle dispersion of Example 24 with various dilutions: 100%, 20%, 5%,1% (from left to right).

FIG. 16A illustrates the photoluminescence spectrum of the polymerparticle dispersion of Example 25 containing TPE-COOH fluorophores.Concentration of the polymer in emulsion: 0.5 wt %; ratio of TPE-COOH topolymer: 0.1%; excitation wavelength: 346 nm.

FIG. 16B illustrates photographs of the polymer nanoparticle emulsion ofExample 25 under normal room illumination (a) and 365 nm irradiationfrom a UV lamp (b).

FIG. 17 illustrates the particle size and size distribution of thefluorescent polymer particles of Example 26. The inset shows thenumber-average diameter (Mean), weight-average diameter (D(3,2)), andcoefficient of variation (C.V.) for the particles.

FIG. 18A is a scanning electron micrograph image of the fluorescentpolymer particles of Example 27 prepared at a surfactant concentrationof 0.

FIG. 18B is a scanning electron micrograph image of the fluorescentpolymer particles of Example 28 prepared at a surfactant concentrationof 0.02 wt %.

FIG. 18C is a scanning electron micrograph image of the fluorescentpolymer particles of Example 29 prepared at a surfactant concentrationof 0.04 wt %.

FIG. 19A is a photograph of the coating film of Example 32 formed by thepolymer nanoparticle dispersion with and without (controls) TPE-COOHfluorophores. The photos were taken under 365 nm irradiation from a UVlamp.

FIG. 19B is a photograph of the flexible thin sheets of Example 33formed by the polymer nanoparticle dispersion with and without(controls) TPE-COOH fluorophores. The photos were taken under 365 nmirradiation from a UV lamp.

FIG. 20A is a transmission electron micrograph image of HeLa celltreated with the fluorescent polymer nanoparticles of Example 34.

FIG. 20B is a transmission electron micrograph image of HeLa cellstreated with the fluorescent polymer nanoparticles of Example 34 uponexcitation of 365 nm UV light.

FIG. 21 is PL spectra of the water/methanol (6:4) solutions of a PPS—OH(5.7×10⁻⁵ M) in the presence of KOH (8.4×10⁻⁴ M) and BSA (atconcentrations given in the figure), as described in Example 35.

FIG. 22 shows the dependency of fluorescence intensity of PPS—OH on BSAconcentration as described in Example 35.

FIG. 23A shows the structure of a G-quartet showing hydrogen bondsbetween G units and interaction with a cation (M⁺).

FIG. 23B shows a G-quadruplex folded by a human telomeric DNA strand.

FIG. 23C shows the sequences of G1 (SEQ ID 1) and its complementary (C1)(SEQ ID 2) and non-complementary (C2) (SEQ ID 3) strands.

FIG. 24 shows the FL spectrum of TPE-SO3 with addition of differentproteins in phosphate buffer saline (pH=7.0). [TPE-SO3]=5 μM. Excitationwavelength: 350 nm.

FIG. 25 shows the 12% Native-PAGE of HSA pre/post-stained by TPE-SO3.The gel is poststained by Coomassie Brilliant Blue for comparison.

FIG. 26A is a plot of quantum yield of TBEPE vs. composition of AN/watermixture.

FIG. 26B shows the FL spectra of TTAPE in a glycerol/water mixture (99:1by volume) at different temperatures; the spectrum of its water solutionat 25° C. is shown for comparison. [dye]=5 μM; ex=350 nm.

FIG. 27A shows the fluorimetric titration of G1 to an aqueous solutionof TTAPE (4.5 μM) in 5 mM Tris-HCl buffer (pH=7.50).

FIG. 27B shows the change in emission intensity (I) at 470 nm withvariation in concentration of G1 or TTAPE; λ_(ex)=350 nm.

FIG. 28 shows the emission spectra of TTAPE in the presence of asolution of G1 (4.5 μM) in 5 mM Tris-HCl buffer (pH=7.50).

FIG. 29 shows the PAGE assays of G1 at concentrations of 0.5, 1.0, 5.0and 10.0 μM (lanes 1-4). The gels were poststained by (A) 10 μM TTAPEand (B) 1.3 μM EB for 5 min.

FIG. 30 is a CD spectra of G1 in a Tris-HCl buffer in the presence orabsence of a metal ion and/or TTAPE at 20° C. [G1]=9 μM, [ion]=0.5 M,[TTAPE]=4.5 μM.

FIG. 31 shows the excitation and emission spectra of TTAPE solutions ina Tris-HCl buffer in the presence or absence of K⁺ ion and/or G1.[TTAPE]=4.5 μM, [G1]=9 μM, [K⁺]=0.5 M; λ_(ex)=350 nm.

FIG. 32A shows the emission spectra of TTAPE in a Tris-HCl buffer in thepresence of G1 and K⁺.

FIG. 32B shows the effects of [K⁺] on emission intensity at 470 nm andpeak wavelength (λ_(em)) of the TTAPE/G1 solution. [TTAPE]=4.5 μM,[G1]=9 μM; λ_(ex)=350 nm.

FIG. 33A shows the effects of addition sequence on FL spectra ofK⁺/TTAPE/G1 complex in 5 mM Tris-HCl buffer solutions. [K⁺]=0.5 M,[TTAPE]=4.5 μM, [G1]=9 μM; λ_(ex)=350 nm.

FIG. 33B shows the effects of addition sequence on CD spectra ofK⁺/TTAPE/G1 complex in 5 mM Tris-HCl buffer solutions. [K⁺]=0.5 M,[TTAPE]=4.5 μM, [G1]=9 μM; λ_(ex)=350 nm.

FIG. 34A shows the dependence of FL intensity of TTAPE at 492 nm oncationic species ([ion]=500 mM). FL spectra of TTAPE in the buffersolutions containing G1 and K⁺ or Na⁺ ion.

FIG. 34B shows the variation in the FL spectrum of TTAPE/G1/K⁺ solutionwith addition of Na⁺ ion. [TTAPE]=4.5 μM, [G1]=9 μM, [K⁺]=100 mM;λ_(ex)=350 nm.

FIG. 35A shows the FL spectra of TTAPE/G1 in a Tris-HCl buffer in thepresence of cationic species. [TTAPE]=4.5 μM, [G1]=9 μM, [ion]=0.5 M;λ_(ex)=350 nm.

FIG. 35B shows the FL spectra of TTAPE in a Tris-HCl buffer in thepresence of cationic species. [TTAPE]=4.5 μM, [G1]=9 μM, [ion]=0.5 M;λ_(ex)=350 nm.

FIG. 36 is a CD spectra of the G1/TTAPE/K⁺ solutions in a Tris-HClbuffer titrated by different amounts of Na⁺ ions. [G1]=9 μM, [TTAPE]=4.5μM, [K⁺]=100 mM; λ_(ex)=350 nm.

FIG. 37A shows the calorimetric curves for titration of G1 in a K-Trisbuffer with serial injections of TTAPE at 25° C.

FIG. 37B shows the binding isotherm as a function of [TTAPE]/[G1] molarratio in the buffer solution.

FIG. 38A shows the emission spectra of buffer solutions (pH=7.50) ofTTAPE/C1 in the absence and presence of metal ions. [TTAPE]=4.5 μM,[ion]=0.5 M; λ_(ex)=350 nm; [C1]=9 μM.

FIG. 38B shows the emission spectra of buffer solutions (pH=7.50) of (A)TTAPE/C1 and (B) TTAPE/G1/C1 in the absence and presence of metal ions.[TTAPE]=4.5 μM, [ion]=0.5 M; λ_(ex)=350 nm; [G1]=[C1]=4.5 μM.

FIG. 39 shows the CD spectra of G1/C1 in the absence or presence of K⁺after hybridization in a Tris-HCl buffer (pH=7.50). [G1]=[C1]=4.5 04, [K⁺]=0.5 M; λ_(ex)=350 nm.

FIG. 40 shows the emission spectra of TTAPE/G1 in a Tris-HCl buffer inthe presence of K⁺ and/or C1 or C2. [TTAPE]=4.5 μM, [K⁺]=0.5 M;λ_(ex)=350 nm; [G1]=9 μM (in the absence of Ci), [G1]=[C1]=4.5 μM (inthe presence of Ci).

FIG. 41 shows the emission spectra of buffer solutions of TTAPE/C2 inthe absence and presence of metal ions. [TTAPE]=4.5 μM, [G1]=[C2]=4.504, [ion]=0.5 M; λ_(ex)=350 nm.

FIG. 42 is the time course of evolution of emission intensity at 470 nmof TTAPE/G1 in a Tris-HCl buffer after injection of a K⁺ solution.[TTAPE]=4.5 μM, [G1]=9 μM, [K⁺]=0.5 M; λ_(ex)=350 nm.

FIG. 43A is the time courses of evolution of emission intensities ofbuffer solutions of TTAPE/G1 after addition of cationic species.[TTAPE]=4.5 μM, [G1]=9 μM; λ_(ex)=350 nm; [K⁺]=0.5 M.

FIG. 43B is the time courses of evolution of emission intensities ofbuffer solutions of TTAPE/G1 after addition of cationic species.[TTAPE]=4.5 μM, [G1]=9 μM; λ_(ex)=350 nm; [Na⁺]=[NH₄ ⁺]=0.5 M,[Ca²⁺]=0.25 M.

FIG. 44A shows the selected crystal structure of G-quadruplex of a humantelomeric DNA (data taken from RSCB Protein Data Bank; ID No. 1KF1).

FIG. 44B shows the molecular structure of TTAPE with minimized energy,simulated by molecular mechanics MM2 program installed in Chem3D Ultra8.0.

FIG. 45 shows the fluorescent bioprobing processes of TTAPE.

FIG. 46A shows the PL spectra of SATPE with/without HSA in the medium ofartificial urine (AU) (pH=6.0)/PBS (pH=7.0). [SATPE]=5 μM; [HSA]=10μg/ml.

FIG. 46B shows the binding isotherm of HSA to SATPE in artificial urinepH=6.0 (black) (plot of FL intensity at 470 nm). The one in PBS pH=7.0(red) is for comparison. [SATPE]=5 μM; excitation wavelength: 350 nm.

FIG. 47A shows the dependence of FL intensity of SATPE at 476 nm ondifferent proteins in phosphate buffer solution. [SATPE]=5 μM,[protein]=100 μg/ml. Excitation wavelength: 350 nm.

FIG. 47B shows the dependence of FL intensity of SATPE at 476 nm ondifferent proteins in artificial urine. [SATPE]=5 μM, [protein]=100μg/ml. Excitation wavelength: 350 nm.

FIG. 48 shows the FL spectrum of SATPE with addition of differentproteins in artificial urine (pH=6.0). [SATPE]=5 μM, [protein]=50 μg/ml.Excitation wavelength: 350 nm.

FIG. 49 is the Synthesis of1,1,2,2-tetrakis[4-(2-triethylammonioethoxy)phenyl]ethene tetrabromide(TTAPE).

FIG. 50 shows the CD spectra of G1 in a Tris-HCl buffer in the presenceor absence of a metal ion and/or TTAPE at 20° C. [G1]=9 μM, [ion]=0.5 M,[TTAPE]=4.5 μM.

FIG. 51 shows the emission spectra of TTAPE/H21 in Na⁺ solution (100 mM)upon K⁺ titration in buffer solutions (pH=7.5). The final concentrationof Na⁺ is kept at 100 mM. [TTAPE]=4.5 μM, [H21]=4.5 μM. Excitationwavelength: 350 nm.

FIG. 52 shows the CD spectra of H21 (5 μM) in Na⁺ solution (100 mM) uponK⁺ titration in 5 mM Tris-HCl (pH=7.50).

FIG. 53 shows the emission spectra of buffer solutions (pH=7.5) ofTTAPE/G1 containing K⁺ and Na⁺ ions. The final concentration of K⁺ iskept at 100 mM. [TTAPE]=4.5 μM, [DNA]=4.5 μM. Excitation wavelength: 350nm.

FIG. 54 shows the CD spectra of buffer solutions (pH=7.50) of TTAPE/G1containing K⁺/Na⁺ ions. The final concentration of K⁺ is kept at 100 mM.[TTAPE]=4.5 μM, [DNA]=9 μM. Excitation wavelength: 350 nm.

FIG. 55 shows the PL spectra of TTAPE (4.5 μM) in the presence ofdifferent concentrations of G-quadruplex DNA (G1 in 150 mM K⁺) in 5 mMTris-HCl buffer (pH=7.5). Excitation wavelength: 350 nm.

FIG. 56 shows the fluorometric titration of G-quadruplex DNA (plot ofthe fluorescence intensity at 470 nm) to the solution with TTAPE (4.5μM) in 5 mM Tris-HCl buffer (pH=7.5) and its fitting curve toOneSiteBind mode. Excitation wavelength: 350 nm.

FIG. 57 shows the PL spectra of 9 μM of G-quadruplex DNA (G1 in 150 mMK⁺) in the presence of different concentrations of TTAPE in 5 mMTris-HCl buffer (pH=7.5). Excitation wavelength: 350 nm.

FIG. 58 shows the job plots for the binding of TTAPE to H21 in Tris-HCl(blue), K-Tris (black), or Na-Tris (red) buffer solution (pH=7.50). Thesum of TTAPE and DNA concentrations was kept at 10 μM. Fluorescentintensities for the bound TTAPE at 470 nm are normalized to the maximumincrease in each case. The y-axis represents the difference in PLintensity for mole fraction of ligand (χ_(L)) in DNA. Intercept molefraction values were determined from least-squares fits to the lineardata portions, giving χ_(int) values of 0.50 (1:1 stoichiometry) for H21in K-Tris, 0.67 (3:1) for H21 in Na-Tris, and 0.75 (4:1) for H21 inTris-HCl, respectively.

FIG. 59 shows the emission spectra of buffer solutions (pH=7.5) ofTTAPE/DNA containing K⁺. [TTAPE]=4.5 μM, [DNA]=4.5 μM. Excitationwavelength: 350 nm.

FIG. 60A shows the emission spectra of TTAPE with different G-rich DNAsequences in the presence of K⁺. [TTAPE]=4.5 μM; [DNA]=9 μM; [K⁺]=0.5 M.Excitation wavelength: 350 nm.

FIG. 60B shows the excitation spectra of TTAPE with different G-rich DNAsequences in the presence of K⁺. [TTAPE]=4.5 μM; [DNA]=9 μM; [K⁺]=0.5 M.Excitation wavelength: 350 nm.

FIG. 60C shows the CD spectra of TTAPE with different G-rich DNAsequences in the presence of K⁺. [TTAPE]=4.5 μM; [DNA]=9 μM; [K⁺]=0.5 M.Excitation wavelength: 350 nm.

FIG. 61 is a photograph of K⁺-Tris-HCl buffer solution of TTAPE in thepresence of different DNAs under the illumination of a handheld UV lamp.[TTAPE]=4.5 μM, [DNA]=4.5 μM, [K⁺]=0.5 M.

FIG. 62A shows the emission spectra of TTAPE/H21 containing K⁺ underenvironments of different pH value. [TTAPE]=4.5 μM, [DNA]=4.5 μM,[K⁺]=0.5 M. Excitation wavelength: 350 nm.

FIG. 62B shows the effect of pH value on the PL intensity at 490 nm ofTTAPE/H21 in the presence of K⁺. [TTAPE]=4.5 μM, [DNA]=4.5 μM, [K⁺]=0.5M. Excitation wavelength: 350 nm.

FIG. 63 shows the prestained poly(acrylamide) gels of (1) DNA ladder onthe left side; (2) H21/TTAPE; (3) H21/Na⁺/TTAPE; (4) H21/K⁺/TTAPE; (5)H21/Ca2⁺/TTAPE; (6) H26/TTAPE; (7) H26/K⁺/TTAPE; (8) H21. On the rightside the figure shows the post-stained gels with either 10 μM TTAPE for10 min.

FIG. 64 shows the FL spectrum of SATPE with addition of HSA inartificial urine solution (pH=6.0). [SATPE]=5 μM. Excitation wavelength:350 nm.

FIG. 65 shows the binding isotherm of BSA to different concentrations ofSATPE in artificial urine (plot of FL intensity at 470 nm). Excitationwavelength: 350 nm.

FIG. 66A shows FL spectra of TPE-SO3 in the presence of native andfibrillar states of bovine insulin.

FIG. 66B shows the change in FL intensity of TPE-SO3 at 470 nm withdifferent concentration of fibrillar (solid circle) and native insulin(open circle). FIG. 66B inset illustrates the FL intensity of TPE-SO3 inan insulin mixture with different molar fractions of fibrillar insulin(f_(f)), where total protein concentration (5 μM) is kept constant ateach run. I₀=FL intensity in the absence of insulin; [TPE-SO3] is 5 μM;λ_(ex)=350 nm.

FIG. 67 shows fluorescence micrographs of insulin fibrils stained byTPE-SO3 solution.

FIG. 68A demonstrates TPE-SO3 FL spectra of insulin incubated atdifferent period of times in buffer solutions (pH 1.6) at 65° C.

FIG. 68B shows TPE-SO3 FL intensity of insulin at 470 nm recorded atdifferent incubation time interval.

FIG. 69A shows effect of incubating insulin with differentconcentrations of TPE-SO3 on insulin fibrillation.

FIG. 69B shows the change in duration of lag phase when incubatinginsulin with increase in TPE-SO3 concentration.

FIG. 69C illustrates the change in fibrillation rate (rF) whenincubating insulin with increase in TPE-SO3 concentration.

FIG. 70 shows MTT toxicity test for TPE-SO3.

FIG. 71 demonstrates the fluorescence behaviour of TPE-SO3 againstvarious proteins.

FIG. 72 shows the emission of TPE-SO3 with amyloid-beta-peptide beforeand after aging.

FIG. 73 shows the rate of fluorescence enhancement of differentconcentrations of TPE-SO3 during lysozyme fibrillation.

FIG. 74 illustrates formation of insulin fibrils under differentconditions: A and D are SEM images and B, C, E and F are TEM imagesshowing insulin after being incubated in the absence (A, B, D and E) andpresence (C and F) of TPE-SO3 in pH 1.6 at 65° C. for 1 hour (A-C) and 7hours (D-F).

FIG. 75 shows effect of TPE-SO3 on fibrillation kinetics of insulin,where TPE-SO3 is added at different time intervals 10 mins (A), 20 mins(B) and 30 mins (C). The dash lines represent the estimated halftimes ofinsulin fibrillation.

FIG. 76 shows changes in FL intensity of TPE-SO3 with native orfibrillar insulin at different pH environments. Dash lines indicate pIof insulin (pH 5.6).

FIG. 77 shows CD spectra of HG21 in Tris-HCl buffer solutions incubatedin the presence of different TPE-derivatives, K⁺ ion or alone at 25° C.[TPE]=4.5 μM; [HG21]=9 μM and [K⁺]=0.5 M.

FIGS. 78A, 78B, 78C, 78D, 78E, and 78F show emission spectra of (A)BSPOTPE, (B) TPE-MitoB, (C) TPE-MitoY, (D) TTAPE-Me, (E) Cy₂Silo, and(F) TPE-ThT, (15 μM) dyed α-syn (5 μM) monomer and fibrils.

FIG. 79 shows peak intensity of BSPOTPE, TPE-MitoB, and TPE-ThT dyedα-syn (5 μM) monomer, oligomer, and fibrils at 470 nm, 480 nm, and 490nm, respectively.

FIG. 80 shows a plot of I/I₀ α-syn (5 μM) fibrillation monitored byTPE-MitoB (15 μM) and TPE-ThT (15 μM).

FIGS. 81A and 81B show the retardation effect of BSPOTPE (50 and 100 μM)on α-syn (5 μM) fibrillation as monitored by 15 μM of TPE-TPP andTPE-ThT, respectively.

FIGS. 82A, 82B, and 82C show fluorescence imaging of α-synuclein fibrilsstained by (A) Cy₂Silo, (B) TPEBe, or (C) TPE-MitoY and Cyanine dye(Cy3).

FIG. 83 shows emission spectra of BSPOTPE (50 μM) monitoring thefibrillation of hIAPP (0.1 mM), λ_(ex)=350 nm.

FIG. 84 shows peak intensity of BSPOTPE (50 μM) dyed hIAPP (0.1 mM)fibril at 470 nm.

FIG. 85 shows emission spectra of BSPOTPE (10 μM) monitoring thefibrillation of hIAPP (0.1 mM) with the presence of DOPC:DOPS (7:3)large unilamellar vesicles (0.9 mM), λ_(ex)=350 nm.

FIG. 86A shows a plot of I/I₀ of BSPOTPE (10 μM) monitoring hIAPP (0.1mM) fibrillation at 380 and 470 nm with increasing incubation time atroom temperature with LUVs.

FIG. 86B shows a plot of the ratio of peak intensity at 380 nm to peakintensity at 470 nm of BSPOTPE (10 μM) monitoring hIAPP (0.1 mM)fibrillation with LUVs at room temperature.

FIG. 87 shows emission spectra of ThT (10 μM) monitoring thefibrillation of hIAPP (0.1 mM) with the presence of DOPC:DOPS (7:3)large unilamellar vesicles (0.9 mM) and AIE luminogens, BSPOTPE andTTAPE-Et, λ_(ex)=430 nm.

FIG. 88 shows a plot of (I-I₀)/(I_(max)-I₀) at 470 nm of BSPOTPE (10 μM)monitored β-amyloid (0.1 mg/mL) fibrillation with different incubationtime.

FIG. 89 shows peak intensity at 470 nm of serial dilution of BSPOTPE (10μM) monitored β-amyloid (0.1 mg/mL) fibrillation with differentincubation time.

FIGS. 90A and 90B show images of β-amyloid fibril (Day 7) with BSPOTPE(50 μM) taken under daylight (A) and UV irradiation (B).

FIG. 91 shows TEM images of β-amyloid fibril at Day 7.

DETAILED DESCRIPTION OF PARTICULAR EMBODIMENTS Definitions

The following definitions are provided for the purpose of understandingthe present subject matter and for constructing the appended patentclaims.

It is noted that, as used in this specification and the appended claims,the singular forms “a,” “an” and “the” include plural references unlessthe context clearly dictates otherwise.

“A chemically conjugated system” means a system of atoms covalentlybonded with alternating single and double bonds in a molecule of anorganic compound.

“A polyene” means a molecule of an organic compound containing more thanone alkene. For example, a diene has two C═C; a triene has three C═C;etc.

“Target molecule” means the molecule whose changes in concentration inan environment are intended to be detected by a sensor. A targetmolecule can comprise or consist of a biomacromolecule. “Detectingmolecule” means a molecule which, upon contacting with a target moleculein the environment, can provide a signal perceivable to human.

“Alkyl” means, unless otherwise specified, an aliphatic hydrocarbongroup which may be straight or branched chain having about 1 to about 15carbon atoms in the chain, optionally substituted by one or more halogenatoms. A particularly suitable alkyl group has from 1 to about 6 carbonatoms. The term “unsaturated” refers to the presence of one or moredouble and triple bonds between atoms of a radical group, for example.

“Heteroatom” means an atom selected from the group consisting nitrogen,oxygen, sulfur, phosphorus, boron and silicon.

“Heteroaryl” as a group or part of a group denotes an optionallysubstituted aromatic monocyclic or multicyclic organic moiety of about 5to about 10 ring members in which at least one ring member is aheteroatom.

“Cycloalkyl” means an optionally substituted non-aromatic monocyclic ormulticyclic ring system of about 3 to about 10 carbon atoms.

“Heterocycloalkyl” means a cycloalkyl group of about 3 to 7 ring membersin which at least one ring member is a heteroatom.

“Aryl” or “aromatic group” means a group or part of a group denotes anoptionally substituted monocyclic or multicyclic aromatic carbocyclicmoiety of about 6 to about 14 carbon atoms, such as phenyl or naphthyl.Examples of substituents of aromatic moiety are those as disclosedherein as R.

“Heteroalkyl” refer to alkyl in which at least one carbon atom isreplaced by a heteroatom.

“Biomacromolecule” means a high molecular biological weight substancecomprising or consisting of one or more of nucleic acids, proteinsand/or complex carbohydrates.

“Alkali metals ions” or “Alkaline earth metals ions” means metalsselected from the group consisting of K⁺, Li⁺, Na⁺, Mg²⁺ and Ca²⁺.

“Microparticle” means any microscopic particle or particle populationhaving a mean diameter of less than about 10 microns (μm); less thanabout 5 μm; less than about 1 μm; or having a mean diameter in the rangeof from greater than or equal to 10 nm to less than 5 μm; of fromgreater than or equal to 40 nm to less than 3 μm; of from greater thanor equal to 50 nm to less than 1 μm; of from greater than or equal to 60nm to less than 750 nm; of from greater than or equal to 50 nm to lessthan 500 nm; of from greater than or equal to 60 nm to less than 300 nm;of from greater than or equal to 80 nm to less than or equal to 250 nm;of from greater than or equal to 1 μm to less than 10 μm; of fromgreater than or equal to 2.5 μm to less than 10 μm; of from greater thanor equal to 5 μm to less than 10 μm; of from greater than or equal to7.5 μm to less than 10 μm; of from greater than or equal to 2.5 μm to7.5 μm; or having a mean diameter in the range of from greater than orequal to 5 μm to 7.5 μm. In an embodiment, greater than 99% of themicroparticles of a microparticle population have a mean diameterfalling within a described range; greater than about 90% of themicroparticles have a mean diameter falling within a described range;greater than about 80% of the microparticles have a mean diameterfalling within a described range; greater than about 70% of themicroparticles have a mean diameter falling within a described range;greater than about 60% of the microparticles have a mean diameterfalling within a described range; greater than about 50% of themicroparticles have a mean diameter falling within a described range;greater than about 40% of the microparticles have a mean diameterfalling within a described range; greater than about 30% of themicroparticles have a mean diameter falling within a described range;greater than about 20% of the microparticles have a mean diameterfalling within a described range; or greater than about 10% of themicroparticles have a mean diameter falling within a described range.

“Nanoparticle” means any microscopic particle or particle populationhaving a mean diameter of less than about 100 nanometers (nm); less thanabout 90 nm; less than about 80 nm; less than about 70 nm; less thanabout 60 nm; less than about 50 nm in diameter; or having a meandiameter of from 1 nm to less than 100 nm; from 10 nm to less than 100nm; from 20 nm to less than 100 nm; from 30 nm to less than 100 nm; from40 nm to less than 100 nm; from 50 nm to less than 100 nm; from 10 nm to90 nm; from 20 to 80 nm; or having a mean diameter of from 30 to 70 nm.In an embodiment, greater than 99% of the nanoparticles of ananoparticle population have a mean diameter falling within a describedrange; greater than about 90% of the microparticles have a mean diameterfalling within a described range; greater than about 80% of themicroparticles have a mean diameter falling within a described range;greater than about 70% of the microparticles have a mean diameterfalling within a described range; greater than about 60% of themicroparticles have a mean diameter falling within a described range;greater than about 50% of the microparticles have a mean diameterfalling within a described range; greater than about 40% of themicroparticles have a mean diameter falling within a described range;greater than about 30% of the microparticles have a mean diameterfalling within a described range; greater than about 20% of themicroparticles have a mean diameter falling within a described range; orgreater than about 10% of the microparticles have a mean diameterfalling within a described range.

“Aggregation-induced emission” or “AIE” means thefluorescence/phosphorescence is turned on upon aggregation formation orin the solid state. When molecularly dissolved, the material isnon-emissive. However, the emission is turned on when the intramolecularrotation is restricted.

“Bathochromic shift” means a change of spectral band position in theabsorption, reflectance, transmittance, or emission spectrum of amolecule to a longer wavelength (lower frequency) due to the influenceof substitution or a change in environment. It is informally referred toas a red shift and is opposite to hypsochromic shift.

“Emission intensity” means the magnitude of fluorescence/phosphorescencenormally obtained from fluorescence spectrometer, fluorescencemicroscopy measurement.

Unless defined otherwise all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which the presently described subject matter pertains.

Where a range of values is provided, for example, concentration ranges,percentage ranges, or ratio ranges, it is understood that eachintervening value, to the tenth of the unit of the lower limit, unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the described subject matter. Theupper and lower limits of these smaller ranges may independently beincluded in the smaller ranges, and such embodiments are alsoencompassed within the described subject matter, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the described subject matter.

Throughout the application, descriptions of various embodiments use“comprising” language; however, it will be understood by one of skill inthe art, that in some specific instances, an embodiment canalternatively be described using the language “consisting essentiallyof” or “consisting of.”

For purposes of better understanding the present teachings and in no waylimiting the scope of the teachings, unless otherwise indicated, allnumbers expressing quantities, percentages or proportions, and othernumerical values used in the specification and claims, are to beunderstood as being modified in all instances by the term “about.”Accordingly, unless indicated to the contrary, the numerical parametersset forth in the following specification and attached claims areapproximations that may vary depending upon the desired propertiessought to be obtained. At the very least, each numerical parametershould at least be construed in light of the number of reportedsignificant digits and by applying ordinary rounding techniques.

Water-Soluble Conjugated Polyenes

Non-limiting examples of water-soluble conjugated polyene functionalAIE-active compounds include the following

R, R′ = —B(OH)₂ X = —(CH₂)_(n)— -(X)_(n)COOR″ —O(CH₂)_(n)— -(X)_(n)COOH—NH(CH₂)_(n)— -(X)_(n)NH₂ —N[(CH₂)_(n)]₂— -(X)_(n)NHR″ —(OCH₂CH₂)_(n)—-(X)_(n)NR″₂ -(X)_(n)N′R″₃Br

-(X)_(n)OH -(X)_(n)SH -(X)_(n)SO₃ ⁻Na⁺

indicates data missing or illegible when filed

In an embodiment, the present subject matter relates to a water-solubleconjugated polyene compound comprising a backbone structure of a formulaselected from the group consisting of:

whereinR and R′ are independently selected from H, X, B(OH)₂, (X)_(n)COOR″,(X)_(n)COOH, (X)_(n)NH₂, (X)_(n)NHR″, (X)_(n)NR″₂, (X)_(n)N⁺R″₃Br⁻,(X)_(n)OH, (X)_(n)SH, (X)_(n)SO₃ ⁻Na⁺, (CH₂)_(n)P⁺R″₃Br⁻,

X is selected from (CH₂)_(n), O(CH₂)_(n), NH(CH₂)_(n), N[(CH₂)_(n)]₂,(CH═CH)_(n) and (OCH₂CH₂)_(n); and R″ is selected from R, R′,(CH₂)_(n)CH₃, CONH—X—, COO—X—, C₆H₅—R, —CH₂—C₆H₅, and C₆H₅; n=0 to 20,and salts thereof, and wherein the compound is water-soluble andexhibits aggregation induced emission.

In another embodiment, the present subject matter relates to awater-soluble conjugated polyene compound, wherein the molecule has abackbone structure of formula I.

In an embodiment, the present subject matter relates to a water-solubleconjugated polyene compound of claim formula I, wherein R is H and R′ isselected from the group consisting of H, OH, COOH, CH₂NH₂, B(OH)₂,O(CH₂)₂SO₃ ⁻Na⁺, O(CH₂)₃SO₃ ⁻Na⁺, O(CH₂)₂N⁺(CH₂CH₃)₃Br⁻,O(CH₂)₂N⁺(CH₃)₃Br⁻, O(CH₂)₄N⁺(CH₂CH₃)₃Br⁻, CH₂PPh₃ ⁺Br⁻,

and N⁺OCH₃.

In a further embodiment, the present subject matter relates to awater-soluble conjugated polyene compound of formula I, wherein R and R′are the same and are selected from the group consisting of OH,O(CH₂)₂N⁺(CH₂CH₃)₃Br⁻, and O(CH₂)₄N⁺(CH₂CH₃)₃Br⁻.

The presently described subject matter is also directed to awater-soluble conjugated polyene compound of formula I, selected fromthe group consisting of

-   1,2-Bis(4-hydroxyphenyl)-1,2-diphenylethylene;-   1,2-Bis(4-methoxyphenyl)-1,2-diphenylethylene;-   1,2-diphenyl-1,2-bis(4,4′-(3-sulfonato)propoxyl)phenylethylene;-   N,N′-[1,2-diphenyl-1,2-bis(1,4-phenoxyethyl)vinyl]bis(triethylammoniumbromide);-   N,N′-[1,2-diphenyl-1,2-bis(1,4-phenoxybutyl)vinyl]bis(triethylammoniumbromide);-   1,1,2,2-tetrakis(4-hydroxyphenyl)ethylene;-   N,N′,N″,N″-[1,2-tetrakis(1,4-phenoxybutyl)vinyl]tetrakis(triethylammoniumbromide);-   N,N′,N″,N′″-[1,2-tetrakis(1,4-phenoxyethyl)vinyl]tetrakis(triethylammoniumbromide);-   4,4′-(1,2-diphenylvinyl)di(phenylboronic acid);-   4,4′-(1,2-diphenylvinyl)di(phenylcarboxylic acid);-   1,2-di[4-(aminomethyl)phenyl]-1,2-diphenylethylene;-   1,2-Bis[4-(3-sulfonatopropoxy)phenyl]-1,2-diphenylethene sodium;-   1,2-Bis[4-(3-triphenylphosphonium)phenyl]-1,2-diphenylethene    bromide;-   1-[4-(1-methyl-4-pyridine)ethene]-1,2,3-triphenylethene    hexafluorophosphate;-   1-[4-(1,2-dimethyl-5-benzothiazole)]-1,2,3-triphenylethene iodide;-   N,N′,N″,N′″-[1,2-tetrakis(1,4-phenoxyethyl)vinyl]tetrakis(trimethylammoniumbromide);    and-   1-[4-(1-ethyl-2-benzothiazole)ethene]-1,2,3-triphenylethene    hexafluorophosphate.

In another embodiment, the present subject matter relates to awater-soluble conjugated polyene compound wherein the molecule has abackbone structure of formula II.

In yet another embodiment, the present subject matter relates to awater-soluble conjugated polyene compound wherein the molecule has abackbone structure of formula III.

In a further embodiment, the present subject matter relates to awater-soluble conjugated polyene compound wherein the molecule has abackbone structure of formula IV.

In an embodiment, the present subject matter relates to a water-solubleconjugated polyene compound of formula IV, wherein R is H and R′ isselected from the group consisting of CH₂N⁺(CH₂CH₃)2Br⁻ andCH₂N(CH₂CH₃)₂.

In another embodiment, the present subject matter relates to awater-soluble conjugated polyene compound of formula IV, selected fromthe group consisting of

-   1,1′-Bis-[4-(N,N′-diethylaminomethyl)phenyl]-2,3,4,5-tetraphenylsilole;    and-   N,N′-[1,1′-bis(1,4-benzylene)-2,3,4,5-tetraphenyl-silolyl)bis(triethylammoniumbromide).

In yet another embodiment, the present subject matter relates to awater-soluble conjugated polyene compound, wherein the molecule has abackbone structure of formula V.

In a further embodiment, the present subject matter relates to awater-soluble conjugated polyene compound, wherein the molecule has abackbone structure of formula VI.

In a further embodiment, the present subject matter relates to awater-soluble conjugated polyene compound, wherein the molecule has abackbone structure of formula VII.

In a further embodiment, the present subject matter relates to awater-soluble conjugated polyene compound, wherein the molecule has abackbone structure of formula VIII.

In a further embodiment, the present subject matter relates to awater-soluble conjugated polyene compound, wherein the molecule has abackbone structure of formula IX.

In a further embodiment, the present subject matter relates to awater-soluble conjugated polyene compound, wherein the molecule has abackbone structure of formula X.

In an embodiment, the present subject matter relates to a water-solubleconjugated polyene compound of claim formula X, wherein R is

In another embodiment, the present subject matter relates to awater-soluble conjugated polyene compound of formula X, which is1,1′-methyl-2,5-bis[4-(1-sulfonatopropyl-2-isoindole)ethenephenyl]-3,4-bisphenylsilole.

Synthesis of Water-Soluble Conjugated Polyenes

In one embodiment, the present subject matter relates to water-solubleconjugated polyenes useful as bioprobes and for manufacturing sensors.These polyenes can be prepared according to a variety of differentmethods. Non-limiting examples of such synthetic methods are discussedbelow.

Appropriately substituted versions of the precursors illustrated in theabove schemes can be readily selected and employed, by the person ofordinary skill in the art to which the presently described subjectmatter pertains, to synthesize corresponding substituted productswithout undue experimentation.

Fluorescent Polymer Particles

The presently described subject matter is directed to water-dispersible,fluorescent, polymeric particles, which comprise or consist of thepresently described TPE-derived water-soluble conjugated polyenes (“TPEdyes”) that exhibit AIE, and a variety of polymer matrices havingdesirable hydrophilicity and chemical composition that can be designedto proved desirable characteristics.

Based on the proposed AIE mechanism, several of the presently describedAIE-active dyes which exhibit fluorescent “turn-on” property when boundto biomacromolecules were investigated. A group of water-soluble AIEmolecules were designed and synthesized. When the presently describedwater-soluble AIE molecules are dissolved in water or phosphate buffersaline (PBS), the solution is virtually non-emissive. However, thefluorescence increases significantly in the presence of proteins andDNA. There is a linear relationship between fluorescent intensity andthe concentration of analytes in a certain range, which is of greatimportance in protein and DNA assays.

Furthermore, the presently described AIE water-soluble molecules areorganic compounds, which make them easily accessible and much moreeconomical compared to platinum or transition metal-containingcounterparts. All of the presently described AIE-active water-solublemolecules are advantageous in that they can be synthesized in manystructural forms and can be easily substituted with a variety offunctional groups.

In addition, the presently described AIE-active water-soluble moleculesare very stable. Virtually no change is observed in theirphotoluminescence spectra when they are stored under ambient temperaturewithout any protection from light and air for more than two months. Thisis distinctly different from other dye molecules, which suffer fromphotobleaching when exposed to room illumination.

In one embodiment, the presently described TPE dyes have conjugatedmolecular structures which can be expressed by the following formula:

wherein R is selected from H, X, B(OH)₂, (X)_(n)COOR″, (X)_(n)COOH,(X)_(n)NH₂, (X)_(n)NHR″, (X)_(n)NR″₂, (X)_(n)N+R″₃Br⁻, (X)_(n)OH,(X)_(n)SH, and (X)_(n)SO₃ ⁻Na⁺;X is selected from (CH₂)_(n), O(CH₂)_(n), NH(CH₂)_(n), N[(CH₂)_(n)]₂,and (OCH₂CH₂)_(n); andR″ is selected from R, R′, (CH₂)_(n)CH₃, CONH—X—, COO—X—, C₆H₅—R,—CH₂—C₆H₅, and C₆H₅; andwherein n=0 to 20.

In an embodiment, R is selected from H, OH, COOH, and NH₂.

In a further embodiment, the present subject matter relates towater-dispersible, fluorescent, polymeric particles, wherein R is H andR′ is selected from H, OH, COOH, and NH₂.

In yet a further embodiment, the present subject matter relates towater-dispersible, fluorescent, polymeric particles, wherein thewater-soluble conjugated polyene compound is selected from the groupconsisting of

-   4,4′-(1,2-diphenylvinyl)di(phenylcarboxylic acid);-   1,2-Bis(4-methoxyphenyl)-1,2-diphenylethylene; and-   1,2-Bis(4-hydroxyphenyl)-1,2-diphenylethylene.

In another embodiment, the present subject matter relates towater-dispersible, fluorescent, polymeric particles, wherein the polymeris a homopolymer or a copolymer comprising one or more monomers selectedfrom the group consisting of a vinylaromatic monomer, an ethylenicmonomer, an alkanoic acid or ester or anhydride, and an ethylchic acidor ester, wherein one or more of the one or more monomers is optionallyfunctionalized.

In a further embodiment, the present subject matter relates towater-dispersible, fluorescent, polymeric particles, comprising at leastone functionalized monomer.

In another embodiment, the present subject matter relates towater-dispersible, fluorescent, polymeric particles, wherein theethylenic monomer is selected from an ethylenic monomer of isoprene,1,3-butadiene, vinylidene chloride, or acrylonitrile; the vinylaromaticmonomer is selected from styrene, bromo-styrene, α-methylstyrene,ethylstyrene, vinyl-toluene, chlorostyrene, chloromethylstyrene, orvinylnaphthalene; the alkanoic acid or ester or anhydride is selectedfrom acrylic acid, methacrylic acid, an alkyl acrylate or an alkylmethacrylate in which the alkyl group possess from 3 to 10 carbon atoms;an hydroxyalkyl acrylate, acrylamide, ethylenic acid ester containing 4or 5 carbon atoms; or a difunctional monomer selected fromdivinylbenzene or 2,2-dimethyl-1,3-propylene diacrylate.

In yet a further embodiment, the present subject matter relates towater-dispersible, fluorescent, polymeric particles, wherein the one ormore monomers are selected from the group consisting of styrene, methylmethacrylate, ethyl acrylate, butyl acrylate, 2-hydroxyethylmethacrylate, acrylic acid, and acrylamide.

In an embodiment, the present subject matter relates towater-dispersible, fluorescent, polymeric particles, wherein the atleast one functionalized monomer is selected from the group consistingof 2-hydroxyethyl methacrylate, 2-aminoethyl methacrylate,trimethylammoniumethyl methacrylate methosulfate, dimethylaminoethylmethacrylate, methacrylic acid, undecylenic acid, methyl propenesulfonic acid, undecylenyl alcohol, oleyl amine, glycidyl methacrylate,acrolein, and glutaraldehyde.

In a further embodiment, the present subject matter relates towater-dispersible, fluorescent, polymeric particles, wherein the one ormore ethylenically unsaturated monomers comprise or consist of methylmethacrylate, butyl acrylate and 2-hydroxyethyl methacrylate.

In another embodiment, the present subject matter relates towater-dispersible, fluorescent, polymeric particles, wherein the methylmethacrylate, butyl acrylate, and 2-hydroxyethyl methacrylate arepresent in a ratio of from 4:5:1 to 5:4:1.

In yet another embodiment, the present subject matter relates towater-dispersible, fluorescent, polymeric particles, wherein the one ormore ethylenically unsaturated monomers comprise or consist of methylmethacrylate, butyl acrylate, and acrylic acid and/or acrylamide.

In a further embodiment, the present subject matter relates towater-dispersible, fluorescent, polymeric particles, wherein the ratioof monomer to functionalized monomer is in the range of from about 3:1to about 20:1.

In another embodiment, the present subject matter relates towater-dispersible, fluorescent, polymeric particles, wherein the ratioof monomer to functionalized monomer is in the range of from about 7:1to about 11:1.

In yet another embodiment, the present subject matter relates towater-dispersible, fluorescent, polymeric particles, wherein the ratioof monomer to functionalized monomer is about 9:1.

In an embodiment, the present subject matter relates towater-dispersible, fluorescent, polymeric particles, having a glasstransition temperature below room temperature.

In a further embodiment, the present subject matter relates towater-dispersible, fluorescent, polymeric particles, comprisingmicroparticles.

In yet a further embodiment, the present subject matter relates towater-dispersible, fluorescent, polymeric particles, wherein themicroparticles comprise a mean particle diameter in the range of fromabout 0.01 μm to about 5 μm.

In another embodiment, the present subject matter relates towater-dispersible, fluorescent, polymeric particles, wherein themicroparticles comprise a mean particle diameter in the range of fromabout 10 nm to about 500 nm.

In an embodiment, the present subject matter relates towater-dispersible, fluorescent, polymeric particles, wherein greaterthan about 50% of the microparticles comprise a mean particle diameterin the range of from about 10 nm to about 500 nm.

In a further embodiment, the present subject matter relates towater-dispersible, fluorescent, polymeric particles, wherein greaterthan about 70% of the microparticles comprise a mean particle diameterin the range of from about 40 nm to about 400 nm.

In yet a further embodiment, the present subject matter relates to amethod for making water-dispersible, fluorescent polymeric particles,comprising or consisting of: dissolving the water-soluble conjugatedpolyene compound in the one or more monomers to form a monomer solution;providing an aqueous composition comprising one or more members selectedfrom the group consisting of a surfactant, a stabilizer and across-linking agent; adding the monomer solution dropwise to the aqueouscomposition to form a mixture; and polymerizing the mixture to producethe water-dispersible, fluorescent, polymeric particles.

In another embodiment, the present subject matter relates to the methodfor making water-dispersible, fluorescent, polymeric particles, whereinpolymerizing comprises emulsion polymerization, microemulsionpolymerization, suspension polymerization, or dispersion polymerization.

In another embodiment, the present subject matter relates to the methodfor making water-dispersible, fluorescent, polymeric particles, whereinthe water-dispersible, fluorescent, polymeric particles are dispersedstably in the aqueous composition.

In a further embodiment, the present subject matter relates to thewater-dispersible, fluorescent, polymeric particles, comprising aformulation selected from a bioprobe, a coating, a paint, a flexiblefree-standing film, a cosmetic, a fluidic tracer, or a marker. A fluidictracer can be used to investigate capillary flow, to define neuronalcell connectivity and to study dye translocation through gap junctions,as well as to follow cell division, cell lysis or liposome fusion. Amarker can be used as an indicator of a biologic state, for example, pH,polarity, and viscosity of the biological environment.

In another embodiment, the present subject matter relates to a flexiblefree-standing film comprising water-dispersible, fluorescent, polymericparticles.

The described TPE dyes possess unique characteristics, in that whenmolecularly dissolved in aqueous solutions, for example, water, emissionis weak, whereas when aggregated in poor non-aqueous solvents orfabricated into thin films, emission is substantially increased.

The TPE dyes can be prepared according to the synthetic routes shown inScheme 2 described herein. Bromo-substituted TPE (TPE-Br) can first beprepared by the McMurry coupling reaction of 4-bromobenzophenone usingtitanium (IV) chloride/zinc as catalyst. Then the bromo groups in TPE-Brcan be transformed into other groups, e.g., carboxyl functionalities, byreaction with n-butyllithium followed by dry ice.

The polymers for encapsulation of the presently described TPE dyes areobtained by polymerization of ethylenically unsaturated monomers. Such apolymer can be a homopolymer or copolymer containing units derived fromvinylaromatic or ethylenic monomers, or from alkanoic or ethyl chicacids or esters, which are optionally functionalized. This type ofpolymer is readily accessible to any person skilled in the art and itwill be sufficient to mention only a few such polymers below, in anon-limiting manner. Such polymers can comprise or consist of one ormore of the following: ethylenic monomers of isoprene, 1,3-butadiene,vinylidene chloride or acrylonitrile type; vinylaromatic monomers suchas styrene, bromo-styrene, alpha-methyl styrene, ethylstyrene,vinyl-toluene, chlorostyrene or chloromethylstyrene, orvinyl-naphthalene; alkanoic acids, esters or anhydrides such as acrylicacid, methacrylic acid, alkyl acrylates and alkyl methacrylates in whichthe alkyl group possesses 3 to 10 carbon atoms; hydroxyalkyl acrylates,acrylamides, ethylenic acid esters containing 4 or 5 carbon atoms; anddifunctional monomers such as divinylbenzene or2,2-dimethyl-1,3-propylene diacrylate and/or other copolymerizablemonomers. Suitable monomers can comprise or consist of styrene, methylmethacrylate, ethyl acrylate, butyl acrylate, 2-hyroxyethylmethacrylate, acrylic acid, and acrylamide. These monomers are usedalone or mixed with each other in any proportion, or alternatively mixedwith another copolymerizable monomer selected from those describedabove. The functional groups can be incorporated onto the surface of thefluorescent particles by, for example, using a mixture of monomer andfunctionalized monomer during the polymerization. The functionalizedmonomer used can comprise or consist of one or more of the following:2-hyroxyethyl methacrylate, 2-aminoethyl methacrylate,trimethylammonium-ethyl methacrylate methosulfate, dimethylaminoethylmethacrylate, methacrylic acid, undecylenic acid, methyl propenesulfonic acid, undecylenyl alcohol, oleyl amine, glycidyl methacrylate,acrolein, glutaraldehyde and the like.

The polymer particles may be formed by the use of appropriatepolymerization techniques such as conventional emulsion polymerization,microemulsion polymerization, suspension polymerization or other meansof polymerization with or without a crosslinking agent such as divinylbenzene or the like. These techniques and agents are well known to thoseof ordinary skill in the art to which the present subject matterpertains. The skilled artisan can readily select and employ suchtechniques and agents without undue experimentation.

The described TPE dyes are dissolved in the monomers prior topolymerization, then incorporated into the polymer matrices through theparticle formation process. TPE dyes are organic in nature, which makesthem readily soluble in the monomers used. TPE dyes can also withstandcommon polymerization conditions. Further, TPE dyes, for example, havingvarious peripheral substituent groups on the aromatic rings, have anaffinity towards the interior of the particles. That is, they arechemically compatible with the polymers constituting the latexparticles. This compatibility is important during the formation of thecorresponding fluorescent polymer particles.

The fluorescent polymer particles prepared according to the presentsubject matter are stable aqueous dispersions whose size is as describedherein and is generally between 0.01 micron and 5 microns, for example,less than 1 micron in diameter, regardless of the polymer composition.The aqueous dispersions have a content of polymer particles from 0.1% to50% by weight relative to the total weight of the dispersion, forexample, from 10% to 30% by weight.

The special characteristics of these fluorescent particles can be variedby incorporating different TPE dyes. The fluorescent intensity of thesepolymer particles can be adjusted by varying the load concentration ofthe TPE dyes. The maximum TPE dye content in the polymer particlesdepends on the nature of the fluorochromes, the encapsulation techniqueused, the nature of the polymer constituting the particles and the sizeof these particles. Load concentration can depend on thefunctionalization density (the number of functional groups per particle)and the interaction (covalent or non-covalent) between the dye and theparticles. The functionalization density depends on the size of theparticles and the nature of the polymer (the polymer chain bearingfunctional groups), while the interaction depends on the nature of thefluorophores, the nature of the polymer, and the encapsulation techniqueused. The nature of the fluorophores depends on the kinds of functionalgroups they are facilitated. The maximum TPE dye content in the polymerparticles may thus vary considerably and reach values of several millionfluorochrome molecules per latex particle. The dye content of thepresently described fluorescent polymer particles can be much higher incomparison to the conventional particles, owing to the absence ofself-quenching of the TPE dyes. On the contrary, the fluorescence of thepresent TPE dyes can be remarkably enhanced at high concentrations,resulting from the AIE effect of TPE dyes.

By using the processes of the present subject matter, fluorescentpolymer particles can be optimized in terms of size, polymercomposition, surface chemistry, and/or spectral characteristics. Thefluorescent polymer particles according to the present subject mattermay be used in all the conventional applications of polymer particleswhich are well known to those skilled in the art (paint, coating,cosmetic, marker, fluidic tracer, etc.). Dye fluorescent polymerparticles according to the present subject matter are more particularlyintended for direct or indirect involvement in biological analyses.

Use of the Conjugated Polyenes

While biosensing processes such as molecular beacons require non-trivialeffort to covalently label or mark biomolecules, presently describedherein is a label-free DNA assay system using a simple dye with AIEcharacteristics as the fluorescent bioprobe. Telomerase is an enzymethat catalyzes the lengthening of telomeres which in turn enables cellsto proliferate. Telomerase is active in 90% of cancer cells and activitythereof is needed for indefinite proliferation. G-quadruplex, asecondary structure of DNA, has been identified in human telomere DNAand such structure is believed to inhibit the activity of telomerase,thereby affects gene expression and controls cancel cell proliferation.TTAPE is non-emissive in solution but becomes highly emissive whenaggregated. When TTAPE is bound to DNA via electrostatic attraction, itsemission is turned on. This process can be reversible. When acompetitive cation is added to the DNA solution, TTAPE is released andits emission is turned off TTAPE works as a sensitive post-stainingagent for electrophoresis gel visualization of DNA. The dye is highlyaffinitive to a secondary structure of G-quadruplex. The bathochromicshift involved in the folding process allows spectral discrimination ofthe G-quadruplex from other DNA structures. The strong affinity of TTAPEdye to G-quadruplex structure is associated with a geometric fit aidedby the electrostatic attraction. The distinct AIE feature of TTAPEenables real-time monitoring of folding process of G1 in the absence ofany pre-attached fluorogenic labels on the DNA strand, TTAPE can be usedas a K⁺ biosensor because its specificity to K⁺-induced and stabilizedquadruplex structure. On the other hand, TPE-SO3, the counterpart ofTTAPE, can serve as a probe for protein detection in aqueous media.TPE-SO3 shows higher affinity to human serum albumin (HSA), the mainprotein in human urine. A higher level of protein loss in the urine,called proteinuria, may mean there is a kidney disease. Thus, TPE-SO3can be potential used for urinary protein detection.

A single-stranded (ss) DNA with guanine (G)-rich repeat sequences canassume a square planar arrangement of the G units stabilized byHoogsteen hydrogen bonds (FIG. 23A). An array of these G-quartets canstack on top of each other to form a secondary structure namedG-quadruplex (FIG. 23B). This structure is further stabilized by themonovalent cations (e.g., K⁺) located in the centers of G-tetrads. It ispredicted that thousands of DNA sequences sprinkled over the humangenome are potential quadruplex forming sites, making the tetradstructure one of the most prevalent regulatory motifs in the body. Mucheffort has been devoted to the studies on the biology of genomic andtelomeric G-quadruplexes. It has been found, for example, thatquadruplex formation can affect gene expression and inhibit telomeraseactivity in cancer cells. It has been envisioned thatquadruplex-targeting drugs may enable artificial regulation of geneexpression and control of cancel cell proliferation. Clearly, efficientformation and stabilization of G-quadruplex structures is a prerequisiteto the rational design of quartet specific medication and telomere-aimedanticancer therapy.

A variety of techniques, including nuclear magnetic resonance (NMR),mass spectroscopy, circular dichroism (CD), UV melting profile analysis,poly(acrylamide) gel electrophoresis (PAGE), and surface plasmonresonance, have been used to study G-quadruplex formation. Thesemethods, however, require large quantities of DNA samples because oftheir poor sensitivities. Fluorescence (FL)-based probe system, on theother hand, offers superb sensitivity, low background noise, and widedynamic working range. A few FL sensors based on “molecular beacons” andfluorescent resonance energy transfer processes have been developed,which prove to be powerful in studying conformational transitions ofquadruplexes, These processes, however, require pre-labeling ofoligonucleotides by fuorophores or dual tagging on a single DNA strandby chemical reactions. Precise synthesis of a DNA-dye conjugate is anontrivial job, and product isolation and purification is oftenpainstaking. In addition, structural changes caused by the chemicalmodifications may affect conformations of the G-quadruplexes andinterfere with their folding kinetics.

Biological processes are undertaking in physiological fluids andaccordingly biological assays are commonly conducted in aqueous buffersolutions. The working units in the FL probes, however, are hydrophobicaromatic rings and other π-conjugated chromophores. The FL dyes tend toaggregate when absorbed onto strand surfaces or after enteringhydrophobic pockets of folded strands, due to the incompatibility oftile dyes with the hydrophilic media and the π-π stacking interactionbetween their π-conjugated chromophores. The aggregate formationnormally quenches light emissions of the dyes, which poses a thornyobstacle to the development of efficient FL probes. Various approacheshave been taken in an effort to impede aggregate formation, such asusing long spacers to separate chromophoric units. Obviously, it isnicer and more desirable to have sensitive and selective G-quadruplexprobes that do not require pre-modifications of tile DNA strands andthat do not suffer from aggregation-caused emission quenching.

A series of non-emissive dyes, such as siloles, butadienes, pyrans,fulvenes, biaryls and TPEs, are induced to luminesce by aggregateformation. The AIE dyes are not only excellent emitters for thefabrication of efficient light-emitting diodes but also sensitive probesfor the detection of biomolecules. Among them, the TPE-based dyes havereceived much attention because of their facile synthesis, readyfunctionalization, good photostability, and high FL quantum yields(φ_(F)).

TTAPE is potentially useful as a G-quadruplex probe, using anoligonucleotide of 5′-GGGTTAGGG-TTAGGGTTAGGG-3′ or [dG₃(T₂AG₃)₃] (G1) asa model DNA that mimics the T₂AG₃ repeat sequences in thesingle-stranded region of a human telomere (FIG. 23C). Other examples ofG-rich repeal sequences which are capable of forming G-quadruplexstructures are disclosed in Hong et al., Chem. Eur. 1. 2010, 16,1232-1245, which is incorporated herein by reference in its entirety. Inan aqueous buffer, the non-emissive TTAPE dye becomes highly luminescentupon its binding to G1 via electrostatic attraction, thanks to itsmultiple positive charges. When G1 folds into G-quadruplex structure,emission peak (λ_(em)) of the AIE dye undergoes a noticeablebathochromic shift, allowing easy differentiation of the G-quadruplexfrom other DNA structures. The folding processes of G1 can be followedby the time-dependent FL measurement of the AIE dye.

In another embodiment, the present subject matter relates to a method ofdetecting G-quadruplex formation, DNA, or protein and protein levelswherein the cation added to the biological sample and polyene mixture isselected from the group consisting of K⁺, Li⁺, Na⁺, NH₄ ⁺, Mg²⁺, andCa²⁺. In a particular embodiment, the cation added to the biologicalsample and polyene mixture is K⁺. In another particular embodiment, thewater-soluble conjugated polyene compound is a TPE, or other compound,as described herein. In this regard, by way of non-limiting example theTPE is selected from the group consisting of TPE-SO3 and TTAPE. Inanother embodiment, the TPE is TTAPE.

In an embodiment, the present subject matter relates to a method ofdiagnosing a kidney disorder comprising contacting a biological samplewith a water-soluble conjugated polyene compound and detectingluminescence. In a particular embodiment, the biological sample isselected from the group consisting of a tissue sample, cell sample,blood, saliva, spinal fluid, lymph fluid, vaginal fluid, seminal fluid,and urine. In another particular embodiment, the biological sample isurine. In a further embodiment, the water-soluble conjugated polyenecompound is a TPE, or other compound, as described herein. In thisregard, by way of non-limiting example the TPE is selected from thegroup consisting of TPE-SO3 and TTAPE. In another embodiment, the TPE isTPE-SO3.

In an embodiment, the present subject matter relates to a method ofscreening a potential anti-cancer drug for activity including contactingsaid anti-cancer drug with a biological sample having a G-rich DNAsequence that is capable of forming a particular G-quadruplex conformerto form a reaction mixture, adding a water-soluble conjugated polyenecompound to the reaction mixture of said anti-cancer drug and saidbiological sample having the G-rich DNA, and detecting luminescence. Inanother embodiment, the water soluble conjugated polyene compound isadded to the biological sample before the anti-cancer drug is mixed withthe biological sample to form the reaction mixture.

In another embodiment, the present subject matter relates to a method ofscreening a potential anti-cancer drug for activity, further comprisingcomparing the detected luminescence from said water-soluble conjugatedpolyene compound in said reaction mixture of said biological sample andsaid anti-cancer drug with a luminescence detected from saidwater-soluble conjugated polyene compound in said biological samplehaving the G-rich DNA sequence alone, wherein a bathochromic shift isobserved in the emission spectrum of said reaction mixture having saidanti-cancer drug and said biological sample to identify theG-quadruplex-inducing ability of the anti-cancer drug.

In a similar embodiment, luminescence is compared with that detected ina biological sample having the G-rich DNA sequence and K⁺ ions alone,wherein the luminescence pattern of said G-rich DNA in the presence ofsaid anti-cancer drug or K⁺ ions are similar. In yet another embodiment,the water-soluble conjugated polyene compound is TTAPE.

In an embodiment, the present subject matter relates to an anti-cancerpharmaceutical composition comprises a water-soluble conjugated polyenecompound. In another embodiment, said compound is conjugated with aG-quadruplex targeting motif. In a further embodiment, said compound isa chemically modified TPE. In another embodiment, said compound is achemically modified TTAPE. In yet another embodiment, one or more of thetriethylamine groups of the TTAPE are substituted with other positivelycharged groups including, piperidine, pyrazole, piperazine andimidazole. The G-quadruplex motif is specific to a DNA sequence which iscapable of forming a particular G-quadruplex conformer in the presenceof said motif. In one embodiment, the G-quadruplex motif in theanti-cancer pharmaceutical composition is isolated from an anti-cancerdrug, which is screened by the present. In another embodiment, theG-quadruplex motif is any of the water-soluble conjugated polyenecompounds disclosed herein which is capable of inducing the formation ofthe particular G-quadruplex conformer.

At certain conditions such as at room temperature, TTAPE also acts as alead compound in an anti-cancer pharmaceutical composition. In oneembodiment, the TTAPE induces the formation of a particular G-quadruplexwhen contacting with a biological sample having a DNA sequence that iscapable of forming a particular G-quadruplex conformer, wherein saidcontacting is carried out at about 25° C. or more.

The present subject matter provides a highly water-soluble AIE-activemolecule capable of discriminating native and fibrillar forms of amyloidproteins and method of use thereof as an external tool for monitoringamyloid fibrillation.

In an embodiment, the present subject matter relates to a method ofmonitoring fibrillation of amyloid protein comprising contacting abiological sample with a water-soluble conjugated polyene compound anddetecting luminescence. In one embodiment, the water-soluble conjugatedpolyene compound is TPE-SO3. In a further embodiment, the contacting iscarried out at a pH value equal to or lower than pH 5.6.

In another embodiment, the present subject matter relates to a method ofmonitoring fibrillation of amyloid protein, wherein the biologicalsample contains amyloid protein which is selected from the groupconsisting of insulin, amyloid beta-peptide, tau, alpha-Synuclein, PrPand polyglutamine-containing proteins or peptides, such as human isletamyloid polypeptide (hIAPP). In yet another embodiment, the amyloidprotein is insulin.

The present subject matter further relates to a method of using thewater-soluble conjugated polyene compound in retarding amyloidfibrillation.

In one embodiment, the present subject matter relates to a method ofretarding amyloid fibrillation for storing and delivery of amyloidprotein comprises storing the amyloid protein with a water-solubleconjugated polyene. In an embodiment, the amyloid protein is stored withthe water-soluble conjugated polyene under a pH of lower or equal to pH5.6. In another embodiment, the amyloid protein is selected from thegroup consisting of insulin, amyloid betapeptide, tau, alpha-Synuclein,PrP and polyglutamine-containing proteins or peptides, such as hIAPP. Ina further embodiment, the amyloid protein is insulin. In yet anotherembodiment, the insulin stored is for diabetes treatment.

In another embodiment, the present subject matter relates to a method ofretarding amyloid fibrillation for storing and delivery of amyloidprotein, wherein the water-soluble conjugated polyene is TPE-SO3.

Dye Synthesis

TTAPE was prepared by the synthetic route shown in FIG. 49.Dehydrobromination of 4,4′-dihydroxybenzophenone (DHBP) with1,2-dibromoethane in the presence of potassium carbonate yields4,4′-bis(2-bromoethoxy)-benzophenone (BBEBP). McMurry coupling of BBEBPproduces 1,1,2,2-tetrakis[4-(2-bromoethoxy}phenyl]ethene (TBEPE), whichis quaternized by triethylamine to furnish a salt of TTAPE. The reactionintermediates and final product were fully characterized byspectroscopic methods, from which satisfactory analysis data wereobtained. TBEPE is completely soluble in chloroform, acetonitrile (AN)and THF, slightly soluble in ethanol and methanol, but totally insolublein water. TTAPE, on the other hand, is soluble in water as well as allthe organic solvents mentioned above, due to the amphiphilic nature ofthe ammonium salt.

AIE Effect

When dissolved in its good solvents at molecular level, TBEPE isvirtually nonluminescent. Addition of poor solvents into its solutionsdramatically boosts its emission efficiency. A dilute AN solution ofTBEPE, for example, emits a faint UV light (FIG. 31). When a largeamount of water (99 vol %) is added, the resultant mixture shows anintense FL spectrum peaked at 479 nm. Since water is a nonsolvent ofTBEPE, its molecules must have aggregated in the aqueous mixture. TBEPEis therefore induced to emit by aggregate formation; in other words, itis AIE active. The mixture is transparent and homogeneous, suggestingthat the dye aggregates suspended in the mixture are nanosized. In thedilute AN solution, the phenyl rings of TBEPE can rotate against itscentral olefinic double bond, which nonradiatively deactivates theexcited state and renders the dye non-emissive. The intramolecularrotations are largely restricted in the nanoaggregates in the An/watermixture. This blocks the non-radiative decay channel of the dye andmakes it highly luminescent.

Changes in the φ_(F) values of TBEPE in the AN/water mixtures withdifferent water contents further confirm its AIE nature. In the ANsolution, TBEPE exhibits a negligibly small φ_(F) value (−0.5%), whichremains almost unchanged till up to ˜60% of water is added (FIG. 26A).Afterward the φ_(F) value starts to increase swiftly. In the AN/watermixtures with lower water fractions, TBEPE is genuinely dissolved, whilein the aqueous mixtures with higher water fractions (>60%), the dyemolecules cluster together due to the deterioration in the solvatingpower of the mixture. When the water fraction is increased to 99 vol %,the aφ_(F) value is increased to −18%, which is ˜35-fold higher thanthat in the pure AN solvent. The absolute φ_(F) values of the aggregatesshould be much higher than the relative ones given in FIG. 26A, if thelight scattering caused by the Mie effect of the nanoaggregatcs is takeninto consideration.

TTAPE is completely soluble in water. Owing to its amphiphilic natureassociated with its quaternary tetraalkylammonium moieties, addition ofAN, THF or methanol into its water solution fails to make the dyemolecules aggregate. As a result, the emissions from TTAPE in all thesemixtures are as weak as that in the pure water solution. However,increasing viscosity and decreasing temperature of the solution of TTAPEcan activate its FL process. As can be seen from FIG. 26B, TTAPE in aviscous glycerol/water mixture at 25° C. emits an intense blue light of464 nm. When the viscous mixture is cooled to ˜78 C., its emissionintensity is further increased. At the cryogenic temperature, solventviscosity is increased and molecular motions are further hampered. TTAPEcan thus be induced to emit by restricting its intramolecular rotations,which is the exact cause for the AIE effect of its TBEPE cousin (videsupra).

DNA Probing

G1 is a 21-mer ssDNA that mimics human telomeric sequence. When the DNAis added into a solution of TTAPE in a Tris-HCl buffer, the solutionstarts to luminesce (FIG. 27A). Similar FL “lightup” phenomenon has beenobserved in the thiazole orange system. The I/I₀ ratio of TTAPE at 470nm increases rapidly in a narrow DNA concentration range and reaches itsmaximum at [G1]=5 μM (FIG. 27B). While conventional FL dyes suffer fromself-quenching problems at high dye concentrations, the FL of TTAPE iscontinuously intensified with increasing its concentration (FIG. 28 andFIG. 27B), thanks to its unique AIE feature. In the aqueous buffer, thecationic dye spontaneously binds to the anionic DNA via electrostaticattraction, resulting in the formation of a TTAPE/G1 complex.Hydrophobic interaction between TTAPE and G1 may have also played a rolein the binding process. These intermolecular forces lock conformationsof the TTAPE molecules bound to the G1 strands. Consequently, theintramolecular rotations of TTAPE are restricted, which thus blocks itsradiationless relaxation pathways and activates its FL process.

The FL “turn-on” switching of TTAPE by binding to G1 strand inspires usto check whether it can be used as a DNA marker in the PAGE assay.Electrophoresis assay of G1 was performed on a Hoefer miniVE system in1×TBE buffer under nondenaturing conditions using a 20% native poly(acrylamide) gel at 100V for 3 h at 40 C. An AlphaDigiDoc™ system with aDE-500 Multilmage™ II light cabinet and an ML-26 UV transilluminator(Alpha Innotech) was used for data collection and analysis. The gel waspoststained with a 10 μM TTAPE solution for 5 min at room temperature,rinsed with distilled water, and photographed under UV light at 290-330nm by the gel documentation system. EB was used to poststain the gel inparallel for comparison. Concentrations of G1 in the range of 0.5-10.0μM were used to check the visualizability of the dye-stained DNA in thePAGE assay.

After running PAGE of G1 in a Tris-borate-ethylene-diaminetetraaceticacid (TBE) buffer, the gel is stained by a TTAPE solution for 5 min.Upon UV illumination, the stained gel shows FL bands at various G1concentrations (FIG. 29A). Ethidium bromide (EB) is a widely usedvisualization agent for PAGE assay. The gel stained by EB exhibitsbright background emissions (FIG. 29B), although a much lower EBconcentration has been used. The G1 bands are not visualized until thegel has been stained by EB for as long as 30 min. Band visualization byEB is usually realized by its intercalation into the hydrophobic regionof DNA, which makes the staining a slow process. On the other hand, theFL of TTAPE is activated by its spontaneous electrostatic interactionwith charged surface of DNA, which can be achieved in a short time.Sensitivity test reveals that TTAPE can detect −0.5 μM of G1. Thedetection limit can be further lowered by increasing tile dyeconcentration, as suggested by tile solution I/I₀ data (c.f., FIG. 27B).The present subject matter thus has the advantages of fast response andhigh sensitivity, in addition to its excellent miscibility with aqueousmedia.

Effects of Cationic Species

Among the cationic species, Na⁺ and NH₄ ⁺ are known to be able to inducequadruplex formation but the TTAPE emission is still diminished by thesecations. It is known that the conformation of G-quadruplex is highlydependent on the type of cationic species. In the presence of Na⁺, G1exhibits a CD spectrum with positively and negatively signed Cottoneffects at 295 and 265 nm, respectively (cf., FIG. 30). This suggeststhe formation of a G-quadruplex with an antiparallel strand alignment,which differs from the G-quadruplex with mixed parallel/antiparallelstrand arrangements fanned in the presence of K⁺. The difference in thegeometric conformation may account for the observed difference in theemission behavior. This offers an attractive possibility of using TTAPEas a bioprobe to discriminate between quadruplexes with differentconformations.

Various electrolytes exist in the biological systems and excess orabsence of one or two of these ionic species can cause biomolecules toundergo conformational transitions, resulting in either favorablebiological effect or undesirable dysfunction. As can be seen from FIG.34B, addition of Na⁺ into TTAPE/G1/K⁺ gradually decreases the intensityof the quadruplex-specific emission at 492 nm. The large amount of Na⁺ions drives the dye molecules chemisorbed on the G-quadruplex surfaceinto the aqueous media, resulting in the observed emission attenuation.The spectral profile, however, remains unchanged, even when the amountof the added Na⁺ ions is 12-fold higher than that of K⁺, indicating thatthe G-quadruplex has maintained its structural integrity. This isfurther proved by the CD data: the CD spectrum of the G-quadruplexformed in the presence of K⁺ is unaffected by the perturbations from theexcess amount of externally added Na⁺ ions (FIG. 36). This suggests thatthe K⁺-containing quadruplex is more stable than the Na⁺ one or that K⁺is superior to Na⁺ in inducing/stabilizing the quadruplex structure.

It is clear that TTAPE is highly affinitive to K⁺-containingG-quadruplex but not its Na⁺ cousin. Isothermal titration calorimetry(ITC) measurements are performed, in an effort to understand thethermodynamic basis for the binding affinity difference. ITC is asensitive technique for the studies of bimolecular processes and canprovide direct information about binding affinities and associatedthermodynamic parameters. A calorimetric titration experiment wasperformed at 25.00±0.01° C. on a MicroCal VP-ITC apparatus. G1 solutionsfor the ITC experiments were prepared in all-potassium (K-Tris: 5 mMTris-HCl and 150 mM KCl) or all-sodium (Na-Tris: 5 mM Tris-HCl and 150mM NaCl) buffer at pH 7.50, as required. The buffer solution of G1 washeated to 85° C. and cooled slowly to ensure the folding of the DNA intoG-quadruplex structure. For a typical titration, a series of 10 μLaliquots of TTAPE solution were injected into the G1/M⁺ solution at a240 s interval. The heat for each injection was determined by theintegration of the peak area in the thermogram with respect to time.Blank titration was conducted by injecting TTAPE into the sample cellcontaining only buffer under the same condition. The interaction heatwas corrected by subtracting the blank heat from that for the TTAPE/G1titration. The k_(b) values were derived by fitting the isotherm curveswith Origin 5.0 software.

As can be seen from FIG. 37A, injection of a tiny aliquot (10 μL) of aTTAPE solution into a G1/K⁺ buffer yields a large exothermic peak.Fitting the data of integrated heat generated per injection in thebinding isotherm (FIG. 37B) gives a binding constant (K_(b)) of 2.4×10⁵M⁻¹, from which a Gibbs energy (ΔG°) of −7.3 kcal mol⁻¹ is obtained.However, in the case of the Na⁺ titration, the binding constant is toosmall to be determined accurately from the ITC data.

Biosensing Processes

It has now become clear that TTAPE is a fluorescent marker that canperform multiple functions, including DNA probing, G-quadruplexrecognition, and potassium-ion sensing (FIG. 45). The non-emissive TTAPEmolecules dissolved in the aqueous buffer become highly luminescent uponbinding to G1 via mainly electrostatic attraction, as the DNA bindingrestricts their intramolecular rotations (Process 1 of FIG. 45).Addition of competitive cations such as Li⁺, Na⁺, NH₄ ⁺, Mg²⁺ and Ca²⁺weakens or quenches the emission, because the cations drive the bounddye molecules back to solution (Process 2 of FIG. 45). Addition of K⁺,however, induces G1 to fold into a G-quadruplex structure, resulting ina red shift in the emission spectrum (Process 3 of FIG. 45).Hybridization with a complementary ssDNA (C1) unfolds the G-quadruplexand affords a duplex. The K⁺ ions in the solution compete with the TTAPEmolecules for binding with the dsDNA. The dye molecules are releasedback to the solution and the emission is thus diminished (Process 4 ofFIG. 45). On the other hand, a noncomplementary ssDNA strand (C2) doesnot disassemble the G-quadruplex structure. As a result, thecharacteristic emission of the quadruplex/TTAPE complex at 492 nm ispreserved (Process 5 of FIG. 45).

TTAPE can function as a “light-up” bioprobe for DNA detection,G-quadruplex identification, and potassium-ion sensing. TTAPE can beutilized as an external fluorescent marker to study conformationalstructures, to monitor folding processes of label-free oligonucleotideswith G-rich strand sequences, and to visualize DNA bands in PAGE assay.The spectral red-shift diagnostically signals the presence of quadruplexstructure, allowing a visual distinguishment of G-quadruplex from otherDNA conformations. Accordingly, TTAPE has application in biomedicinestudies, especially for high-throughput quadruplex-targeting anticancerdrug screening.

Anti-Cancer Therapy

Not only does TTAPE behave as a fluorescence marker, which enables thedetection of G-quadruplex DNA structure, TTAPE also has an effect ininducing G-quadruplex structure formation. Specifically, TTAPE promotesformation of G-quadruplex at about 250° C. or higher. Incubating the DNAsample with TTAPE (TPE-1) at 25° C. results in a spectral profilesimilar to the DNA sample incubated with K⁺ ions at the same temperature(FIG. 77), while incubating the DNA sample with other TPE derivatives(labeled as TPE-2, TPE-3 and TPE-4 in FIG. 77) show a similar spectralprofile as the control (i.e., DNA alone). This G-quadruplex formationability of TTAPE at about 25° C. or higher indicates its application asa lead compound of quadruplex targeting drugs for anti-cancer therapy.In order to be a lead compound in quadruplex-targeting anti-cancerdrugs, TTAPE is chemically modified by substituting one or more of thetriethylamine groups with other positively charged groups includingpiperidine, pyrazole, piperazine and imidazole. Such chemicalmodification does not substantially affect the G-quadruplex-inducingproperty of TTAPE at the above specified temperature. Furthermore,through subsequent structural screening and modification, such asconjugation of the substituted TTAPE to a G-quadruplex targeting motif,an efficient anti-cancer pharmaceutical composition based on targeting aspecific G-rich sequence and/or G-quadruplex can be developed.

Detection of Amyloid Fibril Formation

TPE-SO3 was synthesized according to synthesis scheme 1 herein. The AIEeffect of TPE-SO3 is highly specific and sensitive. As seen in FIG. 66A,the TPE-SO3 solution is non-emissive in the absence of proteins. Thesolution remains faintly luminescent at ca. 470 nm upon addition ofnative bovine insulin (fainted line in FIG. 66A). Native insulin is a51-residue hormone and adopts a primarily helical structure, On theother hand, insulin can favorably form amyloid fibrils in vitro undercertain conditions such as elevated temperature and low pH. Therefore,when the native insulin is heated at high temperature (e.g., 65° C.) andlow pH (e.g. pH 1.6), fibrillar insulin is gradually formed and theTPE-SO3 solution becomes luminescent (solid line in FIG. 66A). Itsemission intensity is enhanced progressively with an increase inconcentration of fibrillar insulin. Close inspection of the bindingisotherm reveals a linear relationship at low insulin concentrationrange (0-5 μM) (FIG. 66B). The fluorescence of TPE-SO3 remainsnegligible even when the concentration of native insulin is varied from0 to 100 μM. Further, the fluorescence emission of TPE-SO3 to fibrillarinsulin is demonstrated to be unaffected in the presence of nativeinsulin (FIG. 66B inset). The emission of TPE-SO3 increasesmonotonically with a linear increase of fibrillar insulin fraction.

TPE-SO3 of the present subject matter has excellent water solubility dueto its sulfonate groups. As a result, TPE-SO3 solutions can be used asdetection tools to stain amyloid fibrils and the stained amyloid fibrilscan be visualized under fluorescence microscope with minimal backgroundfluorescence noise (FIG. 67).

The distinct FL behaviors of TPE-SO3 toward native and fibrillar insulinas well as the highly solubility thereof clearly demonstrate itsusefulness as an external indicator for monitoring the kinetic ofamyloid fibril formation.

Retardation Effect on Amyloid Fibril Formation

TPE-SO3 is surprisingly found to have a retardation effect on amyloidfibril formation. In situ incubation of TPE-SO3 with insulin solutionhas shown to lengthen the lag phase of fibrillation and decelerate theelongation rate of the growth phase in a concentration dependent manner(FIGS. 69A and 69B). The growth rate of insulin fibrillation is alsodecreased exponentially against the concentration of TPE-SO3 (FIG. 69C).These results indicate that TPE-SO3 may interfere with the denaturationof native insulin as well as the intermolecular interactions between theinsulin molecules. Further studies on the retardation effect of TPE-SO3on insulin fibril formation shows that the effect is more significant atthe early stage in the time course of the insulin fibril formation (FIG.75). TPE-SO3 is also shown to be non-toxic; cell viability is unaffectedor hardly influenced in the presence of TPE-SO3 (FIG. 70). TPE-SO3 issafe for administration in combination with pharmaceuticals, thusnon-toxic TPE-SO3 is useful as an amyloid protein stabilizer, such asfor use in long-term storage and delivery of therapeutic insulin indiabetes treatment.

A comparative study of PL (photoluminescence) intensity of TPE-SO3 indifferent amyloid proteins (FIG. 71) shows that TPE-SO3 has the highestselectivity towards insulin fibril over the native insulin and amongother protein monomers. Different forms of amyloid-beta (Aβ) peptide arealso tested with TPE-SO3 (FIG. 72) and the result shows that TPE-SO3 ismore specific to Aβ peptide after aging than before aging. Formation ofAβ fibril is a marker of certain neurodegenerative diseases such asAlzheimer's disease and Parkinson's disease. Another amyloid proteincandidate, lysozyme, which also tends to form fibril under similarphysiological conditions to insulin is also tested with differentTPE-SO3 concentrations in a time course manner (FIG. 73). The resultdemonstrates that the rate of fluorescence enhancement of TPE-SO3 atdifferent concentrations is significantly higher at later stage oflysozyme fibril formation reflecting the exponential growth rate offibrils. The result also demonstrates that the rate of fluorescenceenhancement at each time point increases in a concentration-dependentmanner.

EXAMPLES

The following examples are illustrative of the presently describedsubject matter and are not intended to be limitations thereon.

Example 1 1,2-Bis(4-hydroxyphenyl)-1,2-diphenylethylene (TPE-OH)

A suspension of p-methoxybenzophenone (1.06 g, 5.0 mmol), 1.34 equiv ofTiCl₃/AlCl₃ (5.81 g, 6.7 mmol), and 25 equiv of Zn dust (8.01 g, 122.0mmol) in 100 ml of dry THF was refluxed for 20 h. The reaction mixturewas cooled to room temperature and filtered. The filtrates wereevaporated and the crude product was purified by a silica gel columnusing hexane as eluent. 1,2-Bis(4-methoxyphenyl)-1,2diphenylethene(TPE-OMe) was isolated in 91% yield.

TPE-OMe (1.40 g, 3.56 mmol) was dissolved in 20 ml of dichloromethane(DCM) in a 100 ml flask, and the flask was placed in an acetone-dry icebath at −78° C. A solution of 3.59 g (14.3 mmol) of boron tribromide in10 ml of DCM was added carefully to the mixture under stirring. Theresultant mixture was allowed to warm to room temperature overnightunder stirring. The reaction product was hydrolyzed by careful shakingwith 20 ml of water. The organic phase was separated and concentrated bya rotary evaporator. The crude product was purified by recrystallizationfrom THF/methanol to afford a white solid in 97% yield.

Characterization data of TPE-OMe: ¹H NMR (CDCl₃, 300 MHz) δ (ppm):7.10-7.06 (m, 10H), 6.93 (t, 4H), 6.64 (t, 4H), 3.74 (s, 6H). ¹³C NMR(CDCl₃ 75 MHz) δ (ppm): 158.0, 144.4, 139.7, 136.5, 132.6, 131.5, 127.8,126.3, 113.2, 55.2. MS (TOF) m/e: 392.1 (M+, calcd. 392.2).

TPE-OH: ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 7.11-7.02 (m, 10H), 6.88 (t,4H), 6.56 (d, 4H). ¹³C NMR (CDCl₃, 75 MHz) δ (ppm): 154.1, 144.2, 139.7,135.5, 132.8, 131.5, 127.8, 126.3, 114.7. MS (TOF) m/e: 363.1 [(M−H)+,calcd: 363.1].

Example 2 1,2-Diphenyl-1,2-bis(4,4′-(3-sulfonato)propoxyl)phenylethylene(TPE-SO3/BSPOTPE)

Into a 100 m round-bottom flask were added TPE-OH (0.5 g, 1.37 mmol) and20 m of anhydrous ethanol under nitrogen. The mixture was stirred untilall solids disappeared. A mixture of NaOEt (0.20 g, 3.0 mmol) in 20 mlethanol was added dropwise and stirred for 1 h, causing the colorlesssolution to turn orange-red. Into the solution was added 0.35 g of1,3-propanesultone (2.88 mmol) in 20 m of ethanol. The mixture wasvigorously stirred for 12 h and a white product precipitated out fromthe solution. The product was collected by filtration and washed withethanol and acetone twice to give a white solid in 61% yield.

Characterization data of BSPOTPE: ¹H NMR (DMSO-d6, 300 MHz) δ (ppm):7.25-7.13 (m, 6H), 7.08-7.02 (m, 4H), 6.95-6.90 (m, 4H), 6.81-6.73 (m,4H), 4.09-4.02 (m, 4H), 2.66-2.58 (m, 4H), 2.08-2.02 (m, 4H). ¹³C NMR(DMSO-d6, 75 MHz) δ (ppm): 157.0, 143.9, 139.2, 135.5, 131.9, 130.8,127.8, 126.2, 113.8, 66.4, 47.9, 25.3. MS (TOF) m/e: 631.1 [(M+2H)+—Na,calcd. 631.1], 609.2 [(M+3H)+2Na, calcd. 609.1].

Example 3N,N′-[1,2-Diphenyl-1,2-bis(1,4-phenoxyethyl)vinyl]bis(triethylammoniumbromide) (TPE-C2N+)

To a mixture of sodium hydride (84 mg) and 1,2-bus(4-hydroxyphenyl)-1,2-diphenylethene (0.50 g) in dry dioxane (20 ml),1,2-dibromoethane (1.50 g) was added at room temperature. The mixturewas heated to reflux and stirred for 24 h. After filtration andconcentration, the product was isolated and purified by silica gelchromatography using chloroform/hexane (1:1 v/v) as elute.1,2-Bis[4-(2-bromoethoxy)phenyl]-1,2-diphenylethene (TPE-C2Br) wasobtained in 32% yield.

A 250 ml flask with a magnetic spin bar was charged with TPE-C2Br (100mg) dissolved in 100 ml of THF. To this solution was added triethylamine(5 ml). The mixture was heated to reflux and stirred for 3 days. Duringthis period, 10 ml of water was added at several intervals. THF andextra triethylamine were evaporated. The water solution was washed bychloroform three times. After solvent evaporation, the residue waswashed with chloroform and acetone and then dried overnight in vacuum at50° C. TPE-C2N+ was isolated in 56% yield.

Characterization data of TPE-C2Br: ¹H NMR (300 MHz, CDCl3), δ (ppm):7.10-7.02 (m, 10H), 6.95-6.92 (m, 4H), 6.65-6.59 (m, 4H), 4.15-4.11 (m,4H), 3.55-3.49 (m, 4H). ¹³C NMR (75 MHz, CDCl₃), δ (ppm): 156.6, 144.1,139.8, 137.2, 132.7, 131.5, 127.8, 126.4, 114.0, 67.8, 29.3. MS (TOF),m/e: 578.03 ([M]+, calcd. 578.03).

TPE-C2N+: ¹H NMR (300 MHz, CDCl₃), δ (ppm): 7.09-7.00 (m, 10H),6.97-6.87 (m, 4H), 6.61-6.54 (m, 4H), 3.90-3.84 (m, 4H), 3.45-3.40 (m,4H), 2.00-1.97 (m, 4H), 1.88-1.84 (m, 4H). ¹³C NMR (75 MHz, CDCl₃), δ(ppm): 157.9, 144.9, 140.3, 137.2, 133.2, 132.1, 128.3, 126.9, 114.2,67.3, 34.2, 30.2, 28.6. MS (TOF), m/e: 634.09 ([M]+, calcd. 634.09).

Example 4N,N′-[1,2-Diphenyl-1,2-bis(1,4-phenoxybutyl)vinyl]bis(triethylammoniumbromide) (TPE-C4N+)

The synthesis of the below compound was carried out according to Example3 by using the corresponding dibromobutane.

Characterization data of TPE-C4Br: ¹H NMR (300 MHz, CDCl₃), δ (ppm):7.09-7.00 (m, 10H), 6.97-6.87 (m, 4H), 6.61-6.54 (m, 4H), 3.90-3.84 (m,4H), 3.45-3.40 (m, 4H), 2.00-1.97 (m, 4H), 1.88-1.84 (m, 4H). ¹³C NMR(75 MHz, CDCl₃), δ (ppm): 157.9, 144.9, 140.3, 137.2, 133.2, 132.1,128.3, 126.9, 114.2, 67.3, 34.2, 30.2, 28.6. MS (TOF), m/e: 634.09([M]+, calcd. 634.09).

TPE-C4N+: ¹H NMR (300 MHz, d-DMSO), δ (ppm): 7.25-7.19 (m, 6H),7.07-6.92 (m, 8H), 6.83-6.77 (m, 4H), 4.04-4.02 (m, 4H), 3.36-3.29 (m,16H), 1.86-1.81 (m, 8H), 1.34-1.11 (m, 18H). ¹³C NMR (75 MHz, d-DMSO), δ(ppm): 156.9, 143.8, 139.3, 135.7, 132.0, 130.7, 127.9, 126.4, 113.7,66.5, 55.6, 52.0, 25.5, 18.0, 7.2. MS (TOF), m/e: 789.50 ([M.2H₂O−HBr]+,calcd. 789.44).

Example 5 1,1,2,2-tetrakis(4-hydroxyphenyl)ethylene (DHTPE)

A suspension of 4,4′-dihydroxybenzophenone (3.0 g, 14.0 mmol), 1 equivof TiCl₄ (1.54 ml, 14.0 mmol), and 2 equiv of Zn dust (1.83 g, 28.0mmol) in 100 ml of dry THF was refluxed for 20 h. The reaction mixturewas cooled to room temperature and filtered. The filtrates wereevaporated and the crude product was purified by a silica gel columnusing ethyl acetate (EA) as eluent. DHTPE was obtained as slight yellowpowder of 83% yield.

Characterization data of DHTPE: ¹H NMR (300 MHz, d-DMSO), δ (ppm):9.24-8.94 (br), 7.07-7.04 (d, 4H), 6.95-6.95 (d, 4H), 6.70-6.56 (m, 4H),6.47-6042 (t, 4H). ¹³C NMR (75 MHz, CDCl₃), δ (ppm): MS (FAB), m/e:391.2 ([M−4H]+, calcd. 392.1).

Example 6 1,2-Bis[4-(3-triphenylphosphonium)phenyl]-1,2-diphenylethenebromide (TPE-MitoB)

A suspension of 4-methylbenzophenone (1, 3.6 g, 10.0 mmol), TiCl₄ (1.9g, 10.0 mmol), and Zn dust (1.3 g, 20.0 mmol) in dry THF (100 mL) wasrefluxed for 20 g. Afterward, the reaction mixture was cooled to roomtemperature and filtered. The filtrate was evaporated and the crudeproduct was purified on a silica-gel column using DCM as eluent.1,2-Bis(4-methylphenyl)-1,2-diphenylethene was isolated as white solidin 94% yield. ¹H NMR (300 MHz, CDCl₃), δ (TMS, ppm): 7.11-7.00 (m, 10H),6.91 (d, 8H), 2.26 (d, 6H). δ_(C) (75 MHz, CDCl₃) 144.8, 141.6, 141.1,136.6, 132.0, 131.9, 129.0, 128.2, 126.9, 21.9. m/z (FAB) 360.2 [M⁺];calc. 360.2.

To a mixture of 1,2-Bis(4-methylphenyl)-1,2-diphenylethene (1.8 g, 5.0mmol) and NBS (1.7 g, 10.0 mmol) in CCl₄ was added catalytic amount ofBPO at room temperature. The mixture was stirred and heated to refluxfor 8 h. After filtration and solvent evaporation, the product waspurified by silica gel chromatography using DCM/hexane (1:4 v/v) aseluent. 1,2-Bis[4-(bromomethyl)phenyl]-1,2-diphenylethene was isolatedas pale yellow solid in 43% yield. ¹H NMR (300 MHz, CDCl₃), δ (TMS,ppm): 7.14-7.07 (m, 10H), 7.02-6.96 (m, 8H), 4.41 (d, 4H). ¹³C NMR (75MHz, CDCl₃), δ (TMS, ppm): 144.4, 143.9, 141.5, 136.6, 132.3, 132.0,129.2, 128.5, 127.4, 34.3. m/z (FAB) 518.0 [M⁺]; calc. 518.2.

Triphenylphosphonium salt, TPE-TPP, was prepared from1,2-Bis[4-(bromomethyl)phenyl]-1,2-diphenylethene (0.5 g, 1.0 mmol) andtriphenylphosphine (1.0 g, 4.0 mmol) in DMF at 100° C. After stirringfor 24 h, the solution was poured into large amount of toluene. Thewhite precipitate was collected in 80% yield to obtainBis(Triphenylphosphonium) Tetraphenylethene (TPE-MitoB). ¹H NMR (400MHz, DMSO-d₆), δ (TMS, ppm): 7.90-7.55 (m, 30H), 7.16-6.66 (m, 18H),5.039 (d, 4H). ¹³C NMR (100 MHz, CDCl₃), δ (TMS, ppm): 135.25, 135.05,134.35, 134.31, 134.26, 134.21, 131.06, 130.91, 130.31, 130.18, 130.05,127.72, 126.82, 118.02, 117.17, 34.69. m/z 882.5 [(M−2Br)⁺]; calc.882.4.

Example 7 1-[4-(1-methyl-4-pyridine)ethene]-1,2,3-triphenylethenehexafluorophosphate (TPE-MitoY)

1,4-dimethylpyridinium iodide. 1H NMR (400 MHz, CDCl3), δ (TMS, ppm):8.73 (d, J=6.4 Hz, 2H), 7.92 (d, J=6.0 Hz, 2H), 4.36 (s, 3H), 2.68 (s,3H). HRMS (MALDI-TOF): m/z 107.7845 [(M-I)⁺, calcd 108.0813]

4-(1,2,2-Triphenylvinyl)benzaldehyde. 1H NMR (400 MHz, CDCl3), δ (TMS,ppm): 9.90 (s, 1H), 7.61 (d, J=8.2 Hz, 2H), 7.19 (d, J=8.2 Hz, 2H), 7.11(m, 9H), 7.02 (m, 6H). HRMS (MALDI-TOF): m/z361.1588 [(M+1)⁺, calcd360.1514]

A solution of 4-(1,2,2-Triphenylvinyl)benzaldehyde (200 mg, 0.55 mmol)and iodide salt of 1,4-dimethylpyridinium iodide (130 mg, 0.55 mmol) indry ethanol (15 mL) was refluxed under nitrogen for 48 h. After thereaction mixture was cooled to ambient temperature, the solvent wasevaporated under reduced pressure. The solid was dissolved in acetone (5mL) and a saturated aqueous solution of KPF₆ (5 mL) was then added.After stirring for 30 min, the solution was evaporated to dryness. Theresidue was purified by a silica gel column chromatography usingdichloromethane/acetone mixture (5:1 v/v) as eluent to give a yellowproduct in 53% yield to obtain4-{2-[4-(1,2,2-triphenylvinly)phenyl]vinyl}-1-methylpyridiniumhexafluorophosphate(TPE-MitoY). ¹H NMR (400 MHz, CDCl3), δ (TMS, ppm):8.40 (d, J=6.4 Hz,2H), 7.80 (d, J=5.6 Hz, 2H), 7.51 (d, J=16 Hz, 1H), 7.31 (d, J=8 Hz,2H), 7.13 (m, 19H), 6.95 (d, J=16 Hz, 1H), 4.27 (s, 3H). ¹³C NMR (100MHz, DMSO-d6), δ (ppm): 152.36, 145.45, 145.01, 142.84, 142.68, 141.55,140.06, 139.87, 133.22, 131.37, 130.65, 130.55, 127.91, 127.79, 127.58,126.80, 126.73, 123.37, 123.11, 46.83. HRMS (MALDI-TOF): m/z450.2123[(M−PF₆)⁺, calcd 450.2222].

Example 8 1-[4-(1,2-dimethyl-5-benzothiazole)]-1,2,3-triphenyletheneiodide (TPE-ThT)

A solution of 6-bromo-2-methylbenzothiazole (0.5 mmol, 114 mg),(4-dihydroxyboron-phenyl)-1,2,3-triphenylethene (0.6 mmol, 225.8 mg),sodium carbonate (5 mmol, 530 mg) andTetrakis(triphenylphosphine)palladium (0.03 mmol, 34.7 mg) in THF/H₂O(4:1, v/v) mixture was refluxed for 12 h. Afterward, the resultingmixture was cooled to room temperature and THF was evaporated underreduced pressure. The obtained residue was extracted with CH₂Cl₂ threetimes and the combined organic layer was dried over MgSO4, filtered andthe solvent was evaporated under reduced pressure. The crude product waspurified by silica gel chromatography with hexane/dichloromethane (7:3)as eluent to afford the desired product (215 mg, 0.45 mmol) as yellowsolid. Yield=90%. ¹H NMR (400 MHz, CDCl₃), δ (TMS, ppm): 7.98-7.94 (m,2H), 7.65-7.62 (d, 1H), 7.40-7.38 (d, 2H), 7.14-7.04 (m, 17H), 2.85 (s,3H). HRMS (MALDI-TOF), m/z calcd. for C₃₄H₂₅NS: 479.708. Found 479.11712(M⁺).

To a solution of this product (0.5 mmol, 240 mg) in dry acetonitrile,iodomethane (0.8 mmol, 114 mg) was added dropwise. The reaction mixturewas refluxed for 12 h and cooled to room temperature. The dark solutionwas concentrated under reduced pressure and precipitated in excess ofdiethyl ether. The precipitates were collected and washed by diethylether several times to obtain the desired product (0.23 mmol, 140 mg) asfaint yellow solid. Yield: 45%. ¹H NMR (400 MHz, CDCl₃), δ (TMS, ppm):8.18 (s, 1H), 7.97 (d, 1H), 7.90 (d, 1H), 7.33-7.31 (d, 2H), 7.16-7.03(m, 7H), 4.46 (s, 3H), 3.43 (s, 3H) HRMS (MALDI-TOF), m/z calcd, for(C₃₅H₂₈NS)⁺I⁻: 494.1937. Found 494.1948 (M⁺).

Example 9 1-[4-(1-ethyl-2-benzothiazole)ethene]-1,2,3-triphenylethenehexafluorophosphate (TPEBe)

3-Ethyl-2-methyl-1,3-benzothiazol-3-ium iodide. ¹H NMR (400 MHz, CD3OD),δ (TMS, ppm): 8.33 (d, J=7.8 Hz, 2H), 8.30 (d, J=8.4 Hz, 2H), 7.92 (dd,J1=7.8 Hz, J2=8.4 Hz, 2H), 7.81 (dd, J1=7.8 Hz, J2=8.4 Hz, 2H), 4.85 (q,J=7.5 Hz, 4H), 3.27 (s, 3H), 1.60 (t, 6H, J=7.5 Hz, 6H). HRMS(MALDI-TOF): m/z 178.0660 [(M−I)+, calcd 178.0690]

4-(1,2,2-Triphenylvinyl)benzaldehyde. ¹H NMR (400 MHz, CDCl3), δ (TMS,ppm): 9.90 (s, 1H), 7.61 (d, J=8.2 Hz, 2H), 7.19 (d, J=8.2 Hz, 2H), 7.11(m, 9H), 7.02 (m, 6H). HRMS (MALDI-TOF): m/z 361.1588 [(M+1)⁺, calcd360.1514]

A solution of 4-(1,2,2-Triphenylvinyl)benzaldehyde (200 mg, 0.55 mmol)and iodide salt of 3-Ethyl-2-methyl-1,3-benzothiazol-3-ium iodide (169mg, 0.55 mmol) in dry EtOH (15 mL) was refluxed under nitrogen for 48 h.After cool to ambient temperature, the solvent was evaporated underreduced pressure. The solid was dissolved in acetone (5 mL) and asaturated aqueous solution of KPF₆ (5 mL) was then added. After stirringfor 30 min, the solution was evaporated to dryness. The residue waspurified by a silica gel column chromatography using dichloromethane andacetone mixture (5:1 v/v) as eluent to give a yellow product in 62%yield to obtain3-{2-[4-(1,2,2-triphenylvinly)phenyl]vinyl}-2-methyl-1,3-benzothiazol-3-iumhexafluorophosphate (TPEBe). ¹H NMR (400 MHz, DMSO-d₆), δ (ppm): 8.41(d, J=8.0 Hz, 1H), 8.27 (d, J=8.4 Hz, 1H), 8.13 (d, J=15.6 Hz, 1H), 7.94(d, J=16 Hz 1H), 7.75-7.88 (m 4H), 7.02-7.15 (m 11H), 6.96-7.00 (m 6H),4.91 (q, 2H), 1.42 (t, J=7.2 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d6): δ(ppm): 171.4, 148.4, 147.5, 142.6, 142.4, 142.0, 140.8, 139.6, 131.3,130.6, 130.5, 130.4, 129.5, 129.3, 128.2, 127.8, 127.7, 126.9, 126.7,124.3, 116.5, 112.9, 44.4, 14.0. HRMS (MALDI-TOF): m/z 520.2281[(M−PF₆)⁺, calcd 520.2099]. Anal. Calcd for C₃₇H₃₀F₆NPS: C, 66.76; H,4.54; N, 2.10. Found: C, 67.26; H, 4.45; N, 2.18.

Example 10 N,N′,N″,N′″-[1,2-Tetrakis(1,4-phenoxybutyl)vinyl)tetrakis(triethylammonium bromide) (N+C4-TPE-C4N+)

To a mixture of DHTPE (0.4 g, 20 mmol) and potassium carbonate inacetone, 1,4-dibromobutane (3 ml) was added and the mixture was heatedto reflux and stirred for 24 h. After filtration and concentration, theproduct was isolated and purified by silica gel chromatography usingchloroform/hexane (1:1 v/v) as eluent. The product1,1,2,2-tetrakis(4-(4-bromobutoxy)phenyl)ethane (BrC4-TPE-C4Br) wasobtained as white powder in 21% yield.

Characterization data of BrC4-TPE-C4Br: ¹H NMR (300 MHz, CDCl₃), δ(ppm): 7.08-6.99 (m, 8H), 6.81-6.60 (m, 8H), 3.94-6.86 (m, 8H),3.49-3.42 (m, 8H), 2.07-2.00 (m, 8H), 1.92-1.85 (m, 8H). ¹³C NMR (75MHz, CDCl₃), δ (ppm): 157.2, 136.6, 132.7, 129.5, 114.3, 66.9, 33.9,29.9, 28.3. MS (FAB), m/e: 937.0 ([M]+, calcd. 936.4).

N+C4-TPE-C4N+: ¹H NMR (300 MHz, d-DMSO), δ (ppm): 7.29-7.27 (d, 1H),7.10-7.08 (d, 1H), 6.83-6.80 (d, 7H), 6.83-6.80 (m, 7H), 3.91-3.83 (m,8H), 3.23-3.18 (m, 16H), 2.89-2.84 (m, 8H), 1.70-1.65 (m, 20H),1.17-1.06 (m, 40H). ¹³C NMR (75 MHz, d-DMSO), δ (ppm): 157.3, 135.6,132.6, 130.6, 114.4, 67.2, 56.5, 52.9, 46.5, 26.4, 18.9, 8.0. MS (TOF),m/e: 933.6 ([M−4Br-3CH₂CH₃]+, calcd. 933.7).

Example 11N,N′,N″,N′″-[1,2-Tetrakis(1,4-phenoxyethyl)vinyl)tetrakis(triethylammoniumbromide) (N+C2-TPE-C2N+)

The synthesis of the below compound was carried out according to Example6 by using the corresponding dibromoethane.

Characterization data of BrC2-TPE-C2Br: ¹H NMR (300 MHz, CDCl₃), δ(ppm): 7.71-7.70 (m, 1H), 7.54-7.53 (m, 1H), 7.10-7.07 (d, 4H),6.93-6.90 (d, 3H), 6.84-6.82 (d, 4H), 6.65-6.63 (d, 3H), 4.28-4.22 (m,8H), 3.63-3.60 (m, 8H). ¹³C NMR (75 MHz, CDCl₃), δ (ppm): 156.7, 132.8,130.0, 115.0, 114.1, 68.0, 29.6. MS (TOF), m/e: 823.9 ([M]+, calcd.824.2).

N+C2-TPE-C2N+: ¹H NMR (400 MHz, D₂O), δ (ppm): 6.94-6.89 (m, 8H),6.67-6.65 (m, 4H), 4.24-4.23 (m, 8H), 3.54-3.45 (m, 8H), 3.30-3.25 (m,16H), 3.10-3.05 (m, 8H), 1.20-1.13 (m, 36H). ¹³C NMR (100 MHz, D₂O), δ(ppm): 156.2, 138.2, 133.1, 114.5, 98.0, 61.9, 56.0, 54.2, 54.1, 47.4,9.1, 7.6. MS (TOF), m/e: 1222.5 ([M−2H]+, calcd. 1224.4).

Example 12N,N′,N″,N′″-[1,2-Tetrakis(1,4-phenoxyethyl)vinyl)tetrakis(trimethylammoniumbromide) (TTAPE-Me)

To a mixture of 4,4′-dihydroxybenzophenone (1.0 g, 4.7 mmol) andpotassium carbonate (1.3 g, 9.3 mmol) in acetone (50 mL) was added1,8-dibromooctane (3.8 g, 14.0 mmol). The mixture was refluxed understirring for 12 h. After filtration and solvent evaporation, the crudeproduct was purified by a silica gel column using chloroform as eluent.4,4′-bis(8-bromooctyloxy)benzophenone was obtained as white powder in66% yield (3.10 g). R_(f)=0.5 (chloroform). ¹H NMR (400 MHz, CDCl₃), δ(ppm): 7.78 (d, 4H), 6.94 (d, 4H), 4.05 (t, 4H), 3.42 (t, 4H), 1.89 1.80(m, 8H), 1.48-1.39 (m, 16H). ¹³C NMR (100 MHz, CDCl₃), δ (ppm): 193.9,161.8, 131.6, 129.9, 113.3, 67.5, 33.3, 32.1, 28.5, 28.4, 28.0, 27.4,25.3.

In a suspension of 4,4′-bis(8-bromooctyloxy)benzophenone (1.0 g, 1.7mmol) in 50 mL of THF were added TiCl₄ (0.19 mL, 1.7 mmol) and Zn dust(0.22 g, 3.4 mmol). After refluxing for 20 h, the reaction mixture wascooled to room temperature and filtered. The solvent was evaporatedunder vacuum and the crude product was purified by a silica gel columnusing a chloroform/hexane (1:1 v/v) mixture as eluent.1,1,2,2-tetrakis[4-(8-bromooctyloxy)phenyl]-ethene was obtained asyellow viscous liquid in 62% yield (0.6 g). R_(f)=0.5(chloroform/hexane=1:1). ¹H NMR (400 MHz, CDCl₃), δ (ppm): 6.99-6.90 (m,8H), 6.63-6.60 (m, 8H), 3.87-3.80 (m, 8H), 3.41-3.39 (m, 8H), 1.86-1.70(m, 16H), 1.34-1.26 (m, 32H). ¹³C NMR (100 MHz, CDCl₃), δ (ppm): 157.9,137.5, 133.2, 129.9, 114.2, 68.3, 55.6, 34.7, 33.4, 29.9, 29.3, 28.7,26.6.

1,1,2,2-tetrakis[4-(2-trimethylammonioethoxy)-phenyl]ethene tetrabromide(TTAPE-Me was made by quaternization of1,1,2,2-tetrakis[4-(8-bromooctyloxy)phenyl]-ethene with an excess amountof trimethylamine. Pale yellow powder; 85% yield. ¹H NMR (400 MHz, D₂O),δ (ppm): 7.09 (d, 8H), 6.83 (d, 8H), 4.46 (t, 8H), 3.80 (t, 8H), 3.25(s, 36H). ¹³C NMR (100 MHz, D₂O), δ (ppm): 155.4, 139.2, 137.4, 132.3,113.7, 64.8, 61.6, 53.7. MS (TOF), m/e 855.6561 ([M−2Br−3CH₃]⁺, calcd.855.6725).

Example 13 4,4′-(1,2-diphenylvinyl)di(phenylboronic acid) (TPE-BA)

1,2-bis(4-bromophenyl)-1,2-diphenylethene (0.4 g, 0.82 mmol) wasdissolved in 20 ml of distilled tetrahydrofuran (THF) in a 100 ml flask,and the flask was placed in an acetone-dry ice bath at −78 C. A solutionof 1.0 ml (2.6 mmol) of n-butyllithium (2.5 M in hexane) was addedcarefully to the mixture under stirring. After 1 h, 0.46 ml (4.0 mmol)of trimethyl borate was added to the solution and allowed to react for45 min. The mixture was warmed to room temperature and overnight. Thendilute HCl was used to quench the reaction. After filtration and drying,the product was purified by silica gel column with ethyl acetate aseluent. The product was obtained as yellow solid in 54% yield.

Characterization data of TPE-BA: ¹H NMR (d-MeOH, 300 MHz) δ (ppm):7.26-7.17 (m, 10H), 7.01-6.94 (m, 4H), 6.73-6.65 (m, 4H); ¹³C NMR(d-MeOH, 75 MHz), δ (TMS, ppm): 157.2, 146.2, 141.4, 137.0, 133.9,132.7, 128.9, 127.4, 115.7; MS (TOF) m/e: 422.2 ([M−2H]+ calcd: 420.1).

Example 14 4,4′-(1,2-diphenylvinyl)di(phenylcarboxylic acid) (TPE-CA)

1,2-bis(4-bromophenyl)-1,2-diphenylethene (1 g, 2.04 mmol) was dissolvedin 20 ml of distilled tetrahydrofuran (THF) in a 100 ml flask, and theflask was placed in an acetone-dry ice bath at −78° C. A solution of0.56 ml (6.12 mmol) of n-butyllithium (2.5 M in hexane) was addedcarefully to the mixture under stirring. The solution was transferred toa 500 ml flask with dry ice in it. The resultant mixture was stirredovernight under nitrogen at room temperature. After evaporation of THF,potassium hydroxide solution was added and the aqueous solution waswashed by diethyl ether for several times. 3M hydrochloric acid was usedto acidify the aqueous solution. Ethyl acetate was used to extract theproduct. And the organic layer was dried with MgSO₄ to give the productwith the yield of 24%.

Characterization data of TPE-CA: ¹H NMR (d-Acetone, 300 MHz) δ (ppm):7.99-7.93 (m, 3H), 7.50-7.46 (m, 1H), 7.35-7.28 (m, 9H), 7.25-7.16 (m,4H), 7.15-7.10 (m, 1H); ¹³C NMR (d-Acetone, 75 MHz), δ (TMS, ppm):166.1, 147.8, 142.4, 142.3, 141.1, 132.6, 130.7, 130.5, 128.8, 128.4,127.6, 127.3, 126.7, 126.3, 120.0; MS (TOF) m/e: 403.14 ([M−OH]+ calcd:403.14).

Example 15 1,2-di[4-(aminomethyl)phenyl]-1,2-diphenylethylene (TPE-MA)

A mixture of 1,2-diphenyl-1,2-dip-tolylethene (TPE-Me, 2 g, 5.6 mmol),NBS (2 g, 11.1 mmol) and a catalyst amount of benzoyl peroxide in carbontetrachloride (50 ml) was gently refluxed for 8 h in a 150 mlround-bottom flask. After filtration and concentration, the product wasisolated and purified by silica gel chromatography usingchloroform/hexane (1:4 v/v) as eluent.1,2-bis(4-(bromomethyl)phenyl)-1,2-diphenylethene (TPE-MB) was obtainedas light yellow powder in 45% yield.

A mixture of TPE-MB (0.8 g, 1.5 mmol) and NaN₃ (0.1 g, 1.5 mmol) in DMSO(30 ml) was stirred under N₂ at room temperature for 18 h. The reactionmixture was added to water (200 ml) slowly, and then extracted withdichloromethane. The combined organic layers were dried with MgSO₄ andevaporated to dryness. The crude product was purified by silica gelchromatography eluting with 1:1 chloroform/hexane to give1,2-bis(4-(azidomethyl)phenyl)-1,2diphenylethene (TPE-MN3) as awhite-off solid in 73% yield.

The azido-substituted TPE (TPE-MN3) (0.3 g, 0.7 mmol) was dissolved indry THF (60 ml) and LiAlH₄ (0.15 g, 4.1 mmol) was added slowly at roomtemperature with constant stirring under nitrogen. Following theaddition, the mixture was heated at reflux for 8 h. Water (5 ml-10 ml)was added slowly to decompose the excess LiAlH₄. The solution was thenfiltered and THF was used to wash the solid residue. After evaporationof the organic filtrate, dilute hydrochloric acid was added and theaqueous solution was washed by diethyl ether for several times. Ammoniumhydroxide was used to basify the aqueous solution, following theextraction by diethyl ether. The organic layer was dried with Na₂SO₄ andevaporated to dryness. TPE-MA was obtained as light yellow powder in 87%yield.

Characterization data of TPE-MB: ¹H NMR (CDCl₃, 300 MHz) δ (ppm):7.14-7.07 (m, 10H), 7.02-6.96 (m, 8H), 4.42-4.40 (d, 4H); ¹³C NMR(COCl₃, 75 MHz), δ (TMS, ppm): 144.4, 143.9, 141.5, 126.6, 132.3, 132.0,129.2, 128.5, 127.4, 34.3; MS (TOF) m/e: 518.0 ([M]+ calcd: 518.2).

TPE-MN3: ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 7.12-7.07 (m, 6H), 7.04-6.99(m, 12H), 4.24 (s, 4H); ¹³C NMR (CDCl₃, 75 MHz), δ (TMS, ppm): 144.4,143.9, 141.5, 134.1, 132.4, 131.9, 128.5, 128.4, 127.3, 55.2; MS (TOF)m/e: 400.15 ([M−3N]+ calcd: 400.14).

TPE-MA: ¹H NMR (d-MeOH, 300 MHz) δ (ppm): 7.10-7.06 (m, 10H), 7.00-6.93(m, 8H), 3.75-3.70 (hr, 4H); ¹³C NMR (CDCl₃, 75 MHz), δ (TMS, ppm):143.9, 142.4, 140.7, 131.6, 131.4, 127.8, 127.7, 126.5, 126.4, 30.4; MS(TOF) m/e: 374.1 ([M−NH₂]+ calcd: 374.2)

Example 161,1′-Bis-[4-(N,N′-diethylaminomethyl)phenyl]-2,3,4,5-tetraphenylsilole(A₂-HPS) andN,N′-[1,1′-bis(1,4-benzylene)-2,3,4,5-tetraphenylsilolyl)bis(triethylammoniumbromide) (HPS-(C1N+)2

Into a 500 ml round-bottomed flask were added 150 ml THF, 5 ml water,2.2 g potassium carbonate, 6 ml diethyl amine, and 5 g p-bromobenzylbromide. The resultant mixture was refluxed for 12 h. The mixture wasthen cooled to room temperature, into which 6 ml concentrated HCl wasadded, followed by the addition of 150 ml water. The mixture wasextracted with 100 ml diethyl ether for three times. The diethyl ethersolution was dried with anhydrous magnesium sulfate over night and thenthe magnesium sulfate was removed by filtration. Diethyl ether wasevaporated and the raw product was purified by a silica gel column usinghexane/chloroform mixture (1:1 by volume) as the eluent.(p-bromobenzyl)diethyl amine (BBDA) was obtained in 74% yield (3.6 g).

Into a solution of tolan (5 g, 28 mmol) in THF (25 ml) was added underdry nitrogen lithium shaving (0.214 mg, 31 mmol). The mixture wasstirred for 12 h at room temperature and the resultant green-bluecolored THF solution was added dropwise to a solution oftetrachlorosilane (1.61 ml, 14 mmol) in 125 ml THF. The reaction mixturewas stirred for 2 h at room temperature and then refluxed for 5 h.

Into another flask were added BBDA (6.8 g, 28 mmol) and 80 ml THF. Themixture was cooled to −78° C., into which 10 ml n-BuLi (2.5 M in hexane)was added. After stirring for 1.5 h, the mixture was transferreddropwise at −78° C. to the solution of chlorosilole (preparation shownin previous patent). The reaction mixture was allowed to warm to roomtemperature and was then stirred overnight at that temperature. Then THFwas removed by evaporation, and the crude product was dissolved indiethyl ether. The solution was washed three times by water. The crudeproduct was purified by a silica gel column using chloroform as theeluent at first and changed to ethyl acetate when no by product cameout. The product A₂-HPS was obtained in 28% yield afterrecrystallization from acetone/ethanol mixture.

Characterization data of BBDA: ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 7.40 (d,2H), 7.23 (d, 2H), 3.51 (5,2H), 2.50 (m, 4H), 1.04 (m, 6H).

A₂-HPS: ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 7.56 (d, 2H), 7.32 (d, 2H),7.05-6.75 (m, hr, 20H), 3.55 (s, 4H), 2.54 (m, 8H), 1.04 (m, 12H). ¹³CNMR (75 MHz, CDCl₃), δ (TMS, ppm): 156.6, 142.3, 140.0, 139.9, 139.0,136.2, 130.2, 129.8, 129.0, 127.9, 127.6, 126.5, 125.7, 57.8, 47.2,12.1. MS (CI): m/e calcd. For C₅₀H₅₂N₂Si, 708.4. found 709.4 (M+). UV(THF, 4.0×10⁻⁵ mol/L), λ_(max) (nm): 364. Melting point: 119-120° C.

HPS-(C1N+)₂ was obtained by refluxing A₂-HPS together with bromoethanein acetonitrile.

Example 171,1′-methyl-2,5-bis[4-(1-sulfonatopropyl-2-isoindole)ethenephenyl]-3,4-bisphenylsilole (Cy₂Silole)

To a THF solution of phenylacetylene (4.0 mL, 36.4 mmol) was addedn-BuLi (25.0 mL, 40.1 mmol, 1.6 M solution in hexane) at −78° C. Afterstirring at −78° C. for 4 h, dichlorodimethylsilane (2.2 mL, 18.2 mmol)was added. The mixture was warmed to room temperature and stirredovernight. The solvent was removed under reduced pressure. The mixturewas dissolved in DCM and washed with brine and water. The organic layerwas dried over magnesium sulfate. The crude product was purified by asilica-gel column using hexane as eluent. A colorless solid was obtainedin 86.1% yield. ¹H NMR (400 MHz, CDCl₃), δ (ppm): 7.57 (m, 4H), 7.36 (m,6H), 0.55 (s, 6H). ¹³C NMR (100 MHz, CDCl₃), δ (ppm): 132.1, 128.9,128.2, 122.6, 105.9, 90.6, 0.45. HRMS (MALDI-TOF), m/z 260.1013 (M⁺,calcd 260.1021).

A mixture of lithium (0.056 g, 8 mmol) and naphthalene (1.04 g, 8 mmol)in 8 mL of THF was stirred at room temperature under nitrogen for 3 h toform a deep dark green solution of LiNaph. The viscous solution was thenadded dropwise to a solution of the above product (0.52 g, 2 mmol) in 5mL of THF over 4 min at room temperature. After stirring for 1 h, themixture was cooled to 0° C. and then diluted with 25 mL THF. A blacksuspension was formed upon addition of ZnCl₂.TMEDA (2 g, 8 mmol). Afterstirring for an additional hour at room temperature, a solution of4-bromobenzaldehyde (0.78 g, 4.2 mmol) and PdCl₂(PPh₃)₂ (0.08 g, 0.1mmol) in 25 mL of THF was added. The mixture was refluxed overnight.After cooled to room temperature, 100 mL of H₂O was added and themixture was extracted with DCM. The combined organic layer was washedwith brine and water and then dried over magnesium sulfate. Aftersolvent evaporation under reduced pressure, the residue was purified bya silica-gel column using gradient DCM/hexane (50:50 to 100:0 v/v) aseluent. The product was obtained as a yellow solid in 65.4% yield (0.62g, 1.3 mmol). ¹H NMR (400 MHz, CDCl₃), δ (TMS, ppm): 9.89 (s, 2H), 7.65(d, 4H), 7.10-6.98 (m, 10H), 6.78 (d, 4H), 0.51 (s, 6H). ¹³C NMR (100MHz, CDCl₃), δ (TMS, ppm): 191.7, 155.8, 146.6, 142.2, 137.6, 133.7,129.7, 129.6, 129.1, 127.6, 126.9, −4.1. HRMS (MALDI-TOF): m/z 470.1750(M⁺, calcd 470.1702).

The above compound (0.23 g, 0.48 mmol) was dissolved and refluxed in 25mL of anhydrous ethanol under nitrogen. After it was completelydissolved, 0.53 mL (5.8 mmol) of aniline was added and further refluxedovernight. After cooled by ice water, a lot of precipitates were formedand filtered out. The residue was washed twice by cold ethanol andvacuum-dried without further purification. The product was obtained as ayellow solid in 80.5% yield (0.24 g, 0.39 mmol). ¹H NMR (400 MHz,CDCl₃), δ (TMS, ppm): 8.36 (s, 2H), 7.67 (d, 4H), 7.37 (t, 4H), 7.21 (t,2H), 7.17 (d, 4H), 7.08-6.99 (m, 10H), 6.83 (d, 4H), 0.51 (s, 6H). ¹³CNMR (100 MHz, CDCl₃), δ (TMS, ppm): 160.1, 155.1, 152.2, 143.4, 142.0,138.3, 133.6, 129.9, 129.2, 129.1, 128.6, 127.6, 126.6, 125.8, 120.8,−3.9. HRMS (MALDI-TOF): m/z 621.2725 ([M+H]⁺, calcd 621.2726).

The previous compounds (0.16 g, 0.25 mmol) and (0.18 g, 0.6 mmol) werevacuum-dried and refilled with nitrogen for three times. Afterwards, 25mL freshly distilled THF and 5 ml, of Ac₂O were injected to the mixtureand refluxed for overnight. After cooled to room temperature, thesolvents were evaporated under reduced pressure. The residues wereextracted by chloroform and water. The organic layer was further washedwith brine and water and dried over magnesium sulfate. After solventevaporation, the crude product was purified by a silica-gel column usinggradient chloroform/methanol (v/v from 90:10 to 0:100) mixture aseluent. Red solids Silo-Cy and Silo-2Cy were obtained in 57.9% and 34.5%yields, respectively.

Characterization Data of Cy₂Silo.

¹H NMR (400 MHz, CD₃OD), δ (TMS, ppm): 8.38+8.35 (s+d, 2H), 7.90 (d,4H), 7.87 (m, 2H), 7.75 (m, 2H), 7.68-7.58 (m, 6H), 7.16 (d, 4H),7.10-7.00 (m, 6H), 6.85 (dd, 4H), 4.68 (t, 4H), 2.91 (t, 4H), 2.15 (p,4H), 1.98 (p, 4H), 1.83 (s, 12H), 0.55 (s, 6H). ¹³C NMR (100 MHz,CD₃OD), δ (TMS, ppm): 183.6, 158.0, 156.2, 147.5, 145.2, 144.0, 142.2,139.5, 133.5, 131.8, 131.0, 130.9, 130.6, 128.9, 128.1, 124.1, 116.2,112.5, 53.9, 51.2, 47.8, 28.2, 26.7, 23.3, −4.0. HRMS (MALDI-TOF): m/z1026.4275 ([M+2H]⁺, calcd 1026.4132); 1048.4103 ([M+H+Na]⁺, 1048.3951).

Example 18

Fluorimetric titration of biomacromolecules to polyenes Bovine serumalbumin (BSA) and calf thymus DNA (ctDNA) were selected as modelproteins and DNA. BSA was dissolved in a pH 7.0 phosphate buffersolution (1.0 mg/ml). DNA was dissolved in deionized water (1.0 mg/ml)and filtered through a 0.45 μm filter. The actual concentration (innucleic base) was determined by UV photometry using the extinctioncoefficient ε₂₆₀=6600 M⁻¹ cm⁻¹.

Stock solutions of polyenes were 5×10⁻⁴ M in water. Fluorescencetitration was carried out by sequentially adding 100 μl aliquots of DNAor BSA solutions to a 100 μl stock solution of polyenes, followed byadding an aqueous phosphate buffer (10 mM, pH 7) to acquire a 10.00 mlsolution. The mixtures were stirred for half an hour prior to takingtheir spectra. See FIGS. 1-10 and Table 1 below.

TABLE 1 Photophysical Properties of TPEs in Solution (soln),^(a)Aggregate (aggr),^(b) and Binding (bind)^(c) States λ_(ab), nm^(d) λcm,nm (Φ

 %) ^(e) TPE soln aggr soln aggr bind TPE-OMe 311 330 394 (0.11) 477(15.30) TPE-OH 312 316 393 (0.57) 439 (8.90)  467 (35.7) TPE-SO3 312 320398 (0.37) 442 (17.47) 472 (58.2) ^(a)In acetonitrile for TPE-OMe andTPE-OH (10 μM); in water for TPE-SO3 (5 μM). ^(b)In 99% water/AN mixturefor TPE-OMe and TPE-OH; in 99% AN/water mixture for TPE-SO3. ^(c)In BSAsolution of TPE-OH•Na₂ or TPE-SO3 in an aqueous phosphate buffer with pH= 7.0. ^(d)Absorption maximum. ^(e)Emission maximum (quantum yield givenin the parentheses); excitation wavelength: 350 nm.

indicates data missing or illegible when filed

Example 19

Comparison of water soluble and non-water soluble tetraphenylethylenederivatives in this example, a group of AIE-active TPE derivatives,i.e., derivatives 1-4 below, were synthesized and water-soluble cationicsalts 3 and 4 were evaluated for their utility as bioprobes. In aqueousbuffer solutions, these non-emissive fluorophores become highly emissiveupon binding to protein and DNA molecules through noncovalent, such ashydrophobic and electrostatic, interactions.

Derivative 1: R is —O(CH₂)₂Br Derivative 2: R is —O(CH₂)₄Br Derivative3: R is —O(CH₂)₂N⁺(C₂H₅)₃Br⁻ Derivative 4: R is —O(CH₂)₄N+(C₂H₅)₃Br⁻

The TPE derivatives were prepared by the synthetic route as describedherein. Reactions of 1,2-bis(4-hydroxyphenyl)-1,2-diphenyethene withα,ω-dibromoalkanes in the presence of sodium hydride yielded TPEs 1 and2, whose quaternizations by Net₃ gave salts 3 and 4, respectively.Molecular structures of the TPEs were characterized by spectroscopictechniques, from which satisfactory analysis data were obtained. Dyes 1and 2 are soluble in common organic solvents such as acetonitrile (AN),chloroform and THF but insoluble in water. Salts 3 and 4, on the otherhand, are soluble in water as well as in DMF and DMSO.

Dilute solutions of TPEs 1 and 2 in AN are practically non-luminescent.Addition of non-solvent water into the AN solutions can turn on theemissions of the dyes. From the molecular solution in AN to theaggregate suspension in an AN-water mixture (1:99 by volume), thefluorescent intensity of TPE 1 at 476 nm is increased by −240 fold (FIG.11A). Its absorption maximum shifts from 310 nm in the solution to 330nm in the suspension. The excitation maximum of TPE 1 locates at 330 nm,coinciding well with its absorption maximum. The formation of nanoscopicaggregates of TPE 1 is suggested by the level-off tail in the visibleregion of its absorption spectrum due to the Mie effect of thenanoparticles. Evidently, the emission of TPE 1 is induced by theaggregate formation, or in other words, TPE 1 is AIE-active.

The change of Φ_(F) value of TPE 1 with water fraction in the AN-watermixture further reveals its AIE characteristics (FIG. 11B). In themixtures with water fractions below ˜40%, TPE 1 exhibits negligiblysmall Φ_(F) values (˜0.5%) because the dye molecules are actuallydissolved in the mixtures. The Φ_(F) value of TPE 1 starts to increasewhen the water fraction is increased to ˜50%, at which the solvatingpower of the mixture is decreased to such an extent that the dyemolecules begin to aggregate. The Φ_(F) value reaches ˜20% at a watercontent of 99%, which is ˜40-fold higher than that of its AN solution.The absolute Φ_(F) values of the aggregates should be much higher thanthe relative Φ_(F) values given in FIG. 11B, because the determinationof the latter did not take into consideration the strong absorptioncaused by the Mie effect of the aggregates. TPE 2 exhibits similar AIEbehavior. TPE Salts 3 and 4 are soluble in water. Addition of methanol,AN, THF and dioxane to their water solutions do not cause the salts toaggregate, possibly due to their amphiphilic nature. Their emissions inthe mixtures remain as faint as those in the water solutions. However,increasing the concentrations of the salts can increase their Φ_(F)values, indicating that the salts are also AIE-active.

Complexation of the water-soluble AIE TPEs 3 and 4 with calf thymus DNA(ctDNA) and bovine serum albumin (BSA) were investigated byspectrometric titrations in aqueous phosphate buffer (pH=7.0) at 25 C.Stock solutions of TPEs 3 and 4 (0.25 mM) were prepared. The mixture of100 μl stock solution of 3 with 9.9 ml buffer emits faintly at 395 nmwith a side band at 462 nm. Its absorption maximum locates at 311 nm,with a molar absorptivity of 12400M⁻1 cm⁻¹. Upon addition of the DNA, FLintensity of TPE 3 increased by 5.4 fold. Meanwhile its emission maximumshifted to ˜462 nm, giving a Stokes shift as large as 134 nm. In the DNAconcentration range of 0-100 μg/ml⁻¹, the plot of the FL intensity (1)at 462 nm as a function of DNA concentration (c) is a linear line with acorrelation coefficient of 0.996. Addition of BSA to a buffer solutionof TPE 3 induced a similar effect. The linear range of the I/I_(O)-1 vs.c plot in this case is 0-50 μg/ml⁻¹. The excitation maximums of thesolutions of TPE3 containing BSA and ctDNA both locate at 328 nm.

The effects of the biopolymers on the FL properties of TPE 4 are muchmore pronounced. As can be seen from FIG. 12, I/I_(o) values as high as16.3 and 23.8 are achieved when 300 μg/ml⁻¹ ctDNA and 500 μg/ml⁻¹ BSAare added into solutions of TPE 4, respectively. Clearly TPE 4 is a moresensitive bioprobe. The excitation maximum of TPE 4 is at 328 nm and theStokes shift is ˜135 nm. The linear ranges of TPE 4 are narrower: 0-20μg/ml⁻¹ for DNA and 0-40 μg/ml⁻¹ for BSA. It is clear that the AIE saltsTPEs 3 and 4 can be used as light-up bioprobes for DNA and proteindetection. The probing sensitivity and linear range can be tuned bymodifying their structures.

Regarding the origin of the emission induced by the addition of thebiomacromolecules, the correlation with the AIE nature of the dyes mustbe considered. In both cases, similar shifts in the fluorescent maximums(from 390-399 nm to 463-478 nm) are observed. The excitation spectra ofthe biopolymer-induced emissions are also similar to those of the AIEsfor the TPE derivatives. These facts lead to a natural conclusion thatthe strong blue emissions are from the same excited species.

It appears that the restriction of intramolecular rotations in theaggregates of AIE dyes may have blocked their nonradiative channels,thus making them highly emissive. If the AIE process of the TPE dyesfollows the same mechanism, they should become emissive in the solutionswith high viscosities at low temperatures, because under theseconditions their intramolecular rotations would be hampered. Thefluorescent behaviors of TPE 4 were thus investigated in a highlyviscous glycerol-water (99:1 by volume) mixture at differenttemperatures. At 25° C., the glycerol-water solution of TPE 4 emits astrong blue light of 467 nm with a Stokes shift of 147 nm (FIG. 13),demonstrating that the high viscosity indeed helps. As the solutiontemperature is decreased from 25 to −5° C., the FL intensity of TPE4 isincreased as expected. Its excitation maximum locates at 328 nm, closeto those of its nanoscopic aggregates and its complexes with thebiopolymers.

Dynamic NMR experiments of a dichloromethane solution of 1 reveals thatits resonance peaks are broadened with a decrease in temperature. Theplot of δ_(fwhm) vs. UT gives a linear line, indicating a singlemechanism for the peak broadening. All these results confirm that therestriction of intramolecular rotations plays a crucial role in the AIEprocess. The emissions of the TPE salts can thus be turned on by theaddition of the biomacromolecules. In the buffer solutions containingthe DNA and BSA, the cationic amphiphilic dyes bind to thebiomacromolecules via noncovalent interactions, such as electrostaticattraction (especially for the negative-charged DNA) and hydrophobiceffect (particularly for the protein with hydrophobic pockets in itsnative folding structure). When docked on the surfaces of thebiopolymers and in the cavities of their folding structures, the dyemolecules aggregate with the aid of strong electronic and hydrophobicinteractions between their aryl rings. This suppresses intramolecularrotations of the dye molecules, which in turn impedes theirradiationless transitions and activates their fluorescent processes.Thanks to the AIE nature, the emissions of the TPE-biopolymer complexesare greatly intensified with increasing concentration, for the TPE 4-BSAcomplex. This is truly remarkable, because conventional fluorescentprobes suffer from the ACQ problem at high dye concentrations. Insummary, in this example, AIE active, water-soluble, conjugated polyenecompounds (cationic dyes) have been developed herein for protein and DNAdetection in aqueous media for the first time. The nonemissive dyesolutions become emissive upon addition of the biomacromolecule, forexample, DNA and/or BSA. These AIE compounds exhibit large molarabsorptivities, high quantum yields and wide Stokes shifts and are thusideal “turn-on” fluorescent bioprobes. The restriction of theirintramolecular rotations plays a critical role in their AIE processes.Accordingly, any molecule whose electronic conjugation is affected bythe twisting of multiple pendants around its core due to involved stericeffects can be AIE active. This example demonstrates that AIEluminophors can be utilized as fluorescent probes in the area ofbiological research.

Example 20 Synthesis of 4,4′-(1,2-diphenylvinyl)di(phenylcarboxylicacid) (TPE-COOH)

The scheme of the synthesis of TPE-COOH is shown in Scheme 1 above.1,2-bis(4-bromophenyl)-1,2-diphenylethene (1 g, 2.04 mmol) was dissolvedin 20 ml of distilled tetrahydrofuran (THF) in a 100 ml flask, and theflask was placed in an acetone/dry ice bath at −78 C. A solution of 0.56ml (6.12 mmol) of n-butyllithium (2.5 M in hexane) was added carefullyto the mixture under stirring. The solution was transferred to a 500 mlflask with dry ice in it. The resultant mixture was stirred overnightunder nitrogen at room temperature. After evaporation of THF, potassiumhydroxide solution was added and the aqueous solution was washed bydiethyl ether for several times. 3 M hydrochloric acid was used toacidify the aqueous solution. Ethyl acetate was used to extract theproduct. And the organic layer was dried with MgSO₄ to give the productwith the yield of 24%.

Characterization data of TPE-COOH: ¹H NMR (d-Acetone, 300 MHz) δ (ppm):7.99-7.93 (m, 3H), 7.50-7.46 (m, 1H), 7.35-7.28 (m, 9H), 7.25-7.16 (m,4H), 7.15-7.10 (m, 1H); ¹³C NMR (d-Acetone, 75 MHz), δ (TMS, ppm):166.1, 147.8, 142.4, 142.3, 141.1, 132.6, 130.7, 130.5, 128.8, 128.4,127.6, 127.3, 126.7, 126.3, 120.0; MS (TOF) m/e: 403.14 ([M−OH]+ calcd:403.14).

The absorption and photoluminescence spectra of the dye inacetonitrile/water mixture (1:99 v/v) are shown in FIG. 14. When it ismolecularly dissolved in acetonitrile, it is practically nonfluorescent.However, when large amount of water (insoluble to TPE-COOH yet misciblewith acetonitrile) is added, bright cyan light (˜480 nm) is observed.The emission becomes stronger with an increase in water content,suggesting that TPE-COOH is AIE-active.

Example 21 Synthesis of 1,2-Bis(4-hydroxyphenyl)-1,2-diphenylethylene(TPE-OH)

A suspension of p-methoxybenzophenone (1.06 g, 5.0 mmol), 1.34 equiv ofTiCl₃/AlCl₃ (5.81 g, 6.7 mmol), and 25 equiv of Zn dust (8.01 g, 122.0mmol) in 100 ml of dry THF was refluxed for 20 h. The reaction mixturewas cooled to room temperature and filtered. The filtrates wereevaporated and the crude product was purified by a silica gel columnusing hexane as eluent. 1,2-Bis(4-methoxyphenyl)-1,2diphenylethene(TPE-OMe) was isolated in 91% yield.

TPE-OMe (1.40 g, 3.56 mmol) was dissolved in 20 ml of dichloromethane(OCM) in a 100 ml flask, and the flask was placed in an acetone-dry icebath at −78° C. A solution of 3.59 g (14.3 mmol) of boron tribromide in10 ml of DCM was added carefully to the mixture under stirring. Theresultant mixture was allowed to warm to room temperature overnightunder stirring. The reaction product was hydrolyzed by careful shakingwith 20 ml of water. The organic phase was separated and concentrated bya rotary evaporator. The crude product was purified by recrystallizationfrom THF/methanol to afford a white solid in 97% yield.

Characterization data of TPE-OMe: ¹H NMR (CDCl₃, 300 MHz) δ (ppm):7.10-7.06 (m, 10H), 6.93 (t, 4H), 6.64 (t, 4H), 3.74 (s, 6H). ¹³C NMR(CDCl₃, 75 MHz) δ (ppm): 158.0, 144.4, 139.7, 136.5, 132.6, 131.5,127.8, 126.3, 113.2, 55.2. MS (TOF) m/e: 392.1 (M+, calcd. 392.2).

Characterization data of TPE-OH: ¹H NMR (COCl₃, 300 MHz) δ (ppm):7.11-7.02 (m, 10H), 6.88 (t, 4H), 6.56 (d, 4H). ¹³C NMR (CDCl₃, 75 MHz)δ (ppm): 154.1, 144.2, 139.7, 135.5, 132.8, 131.5, 127.8, 126.3, 114.7.MS (TOF) m/e: 363.1 [(M−H)+, calcd: 363.1].

Example 22N,N′,N″,N′″-[1,2-Tetrakis(1,4-phenoxyethyl)vinyl]tetrakis(triethylammoniumbromide) (N+C2-TPE-C2N+)

THF (Labscan) was purified by distillation from sodium benzophenoneketyl under nitrogen immediately prior to use. DHBP, titanium(IV)chloride, zinc dust, 1,2-dibromoethane, potassium carbonate, acetone,triethylamine, and other reagents were all purchased from Aldrich andused as received.

¹H and ¹³C NMR spectra were measured on a Bruker ARX 300 spectrometerwith tetramethylsilane (TMS; δ=0) as the internal standard. Mass spectrawere recorded on a Finnigan TSQ 7000 triple quadrupole spectrometeroperating in a MALDI-TOF mode. UV spectra were measured on a Milton RoySpectronic 3000 Array spectrophotometer and FL spectra were recorded ona Perkin-Elmer LS 55 spectrofluorometer with a Xenon discharge lampexcitation. Time dependent FL signals were measured using aFluostarOptima multifunctional microplate reader (BMG Labtechnologies)with excitation/emission wavelengths set at 350/470 nm. CD spectra wererecorded on a Jasco J-810 spectropolarimeter in 1 mm quartz cuvetteusing a step resolution of 0.2 nm, a scan speed of 100 nm/min, asensitivity of 0.1°, and a response time of 0.5 s. Each spectrum was theaverage of three scans.

The synthetic route to TTAPE is shown in FIG. 49. McMurry coupling ofBBEBP yields TBEPE, quaternization of which by triethylamine generatesTTAPE. Detailed experimental procedures for the dye synthesis are givenbelow.

To a mixture of DHBP (3.0 g, 14.0 mmol) and potassium carbonate (5.0 g,36.2 mmol) in acetone (50 ml) was added 1,2-dibromoethane (4 ml, 46.4mmol). The mixture was refluxed under stirring for 24 h. Afterfiltration and solvent evaporation, the crude product was purified by asilica gel column using chloroform as eluent. BBEBP was obtained aswhite powder in 70% yield (4.20 g). R_(f)=0.6 (chloroform); ¹H NMR (300MHz, CDCl₃, 25° C., TMS): δ=3.68 (t, 4H; BrCH₂), 4.38 (t, 4H; OCH₂),6.97 (d, 4H, J 9.0 Hz; Ar), 7.78 ppm (d, 4H, J=9.0 Hz; Ar); ¹³C NMR (75MHz, CDCl₃, 25° C., TMS): δ=29.3, 68.6, 114.8, 115.8, 131.9, 133.0,162.0 ppm.

In a suspension of BBEBP (1.0 g, 2.3 mmol) in 50 ml of THF were addedTiCl₄ (0.26 ml, 2.3 mmol) and Zn dust (0.31 g, 4.6 mmol). Afterrefluxing for 20 h, the reaction mixture was cooled to room temperatureand filtered. The solvent was evaporated under vacuum and the crudeproduct was purified by a silica gel column using a chloroform/hexane(1:4 v/v) mixture as eluent. TBEPE was obtained as white solid in 63%yield (0.606 g). R_(f)=0.7 (chloroform/hexane=1:4); ¹H NMR (300 MHz,CDCl₃, 25° C., TMS): δ=3.63 (t, 8H; BrCH₂), 4.23 (t, 8H; OCH₂), 6.66 (d,8H, J=8.7 HZ; Ar); 6.93 ppm (d, 4H, J=8.7 Hz; Ar); ¹³C NMR (75 MHz,CDCl₃, 25° C., TMS): δ=29.8, 68.3, 114.5, 133.3, 138.0, 139.1, 157.0ppm; MALDI-TOF-MS m/z: calcd for C₃₄H₃₂Br₄O₄ ⁺: 823.8993. found 823.8688([M]+).

In a 250 ml flask with a magnetic stirrer was dissolved TBEPE (100 mg,0.12 mmol) in THF (100 ml). After adding an excess amount oftriethylamine (5 ml, 35.6 mmol), the solution was refluxed for 3 days.During the period, 10 ml of water was added at several intervals. Theorganic solvents were evaporated under reduced pressure and the aqueoussolution was washed with chloroform three times. After solventevaporation and drying overnight in vacuo at 50° C., TTAPE was isolatedas yellow viscous liquid in 56% yield (0.089 g). ¹H NMR (400 MHz, D₂O,25° C., TMS): δ=1.13-1.20 (m, 36H; NCH₂CH₃), 3.25-3.30 (m, 24H;NCH₂CH₃), 3.45-3.54 (m, 8H; OCH₂CH₂N), 4.23-4.24 (m, 8H; OCH₂CH₂N),6.65-6.67 (m, 8H; Ar), 6.89-6.94 ppm (m, 8H; Ar); ¹³C NMR (75 MHz, D₂O,25° C., TMS): δ=7.4, 47.2, 54.2, 61.8, 114.5, 133.0, 138.0, 139.5, 156.2ppm; MALDITOF-MS m/z: calcd for C₅₈H₉₂Br₄N₄O₄ [M−2Br]⁺: 1066.5485. found1066.5359 ([M−2Br]⁺).

Example 23 Sodium1,2-bis[4-(3-sulfonatopropoxyl)phenyl]-1,2-dicyanoethene (3)

To prepare 2,3-bis(4-methoxyphenyl)fumaronitrile (1),4-methoxyphenylacetonitrile (3.679 g, 25 mmol) and iodine (6.345 g, 25mmol) were dissolved in 100 mL diethyl ether under nitrogen atmospherein a 250 mL three-necked round bottom flask. The solution mixture wascooled to −78° C. Sodium methoxide (2.836 g, 52.5 mmol) in 30 mLmethanol was added into the solution mixture dropwise over half an hour.The resulting mixture was warmed to 0° C. for 4 hrs and quenched by 75mL 3% v/v HCl solution. The precipitate was filtered and washed withwater, sodium meta bisulfite, water and ethanol respectively. The crudeproduct was purified by recrystallization by DCM and ethanol to yieldgreenish yellow crystal. ¹H NMR (CDCl₃, 300 MHz), (TMS, ppm): 3.885 (s,6H), 6.988-7.039 (d, 4H), 7.770-7.820 (d, 4H). ¹³C NMR (CDCl₃, 75 MHz),(TMS, ppm): 56.2, 115.3, 118.0, 123.4, 125.3, 131.1, 162.7.

To prepare sodium1,2-bis[4-(3-sulfonatopropoxyl)phenyl]-1,2-dicyanoethene (3), (1)(0.2903 g, 1 mmol) was dissolved in 10 mL distilled DCM under N₂atmosphere in a 50 mL two-valves round bottom flask. Boron trifluoridemethyl sulfide complex (5.26 mL, 50 mmol) was added into the solutionslowly. The resulting mixture was stirred at ambient temperatureovernight. The solution was concentrated under a stream of N₂ andpartitioned between 1 M HCl and ethyl acetate. The combined organiclater were washed with H₂O and brine, dried over Na₂SO₄ and concentratedto give 2,3-bis(4-hydroxyphenyl)fumaronitrile (2) without furtherpurification.

Freshly prepared (2) was then dissolved in 20 mL anhydrous ethanol underN₂ atmosphere in a 100 mL three necked round bottom flask. A solution ofsodium ethanoxide (0.2 g, 3.0 mmol) in 20 mL ethanol was added into themixture dropwise over half an hour. The orange red solution was stirredfor 1 hr at room temperature. 1,3 propansultone (0.35 g, 2.9 mmol) in 20mL ethanol was added into the mixture and stirred 12 hrs. Theprecipitate was filtered and washed with ethanol. The crude product waspurified by recrystallization using water and acetone. ¹H NMR (D20, 300MHz), (TMS, ppm): 2.15-2.25 (m, 4H), 2.94-3.07 (m, 4H), 4.10-4.14 (m,4H), 7.02-7.04 (d, 4H), 7.67-7.76 (d, 4H). ¹³C NMR (D₂O, 75 MHz), (TMS,ppm): 24.2, 47.8, 66.7, 115.2, 117.4, 122.8, 124.5, 130.5, 160.8.

Example 24 Preparation of Fluorescent Polymer Particles

Into a 50 ml dropping funnel was dissolved 0.1 wt % TPE-COOH in amonomer mixture of methyl methacrylate, butyl acrylate and2-hydroxyethylmethacrylate, with the volume ratio of 4:5:1. The solutionwas purged with nitrogen for 20 min and then added dropwise into thedeionized water containing the emulsifier sodium dodecyl sulfate (0.2 wt%). The emulsion copolymerization proceeds at 75° C. under 400 rpmagitation for 6-10 h then stops by cooling.

The polymer particle dispersion prepared is quite uniform and stable.The particle dispersion is highly emissive under UV irradiation, even invery dilute state (FIG. 15). It is worthy of noting that the emission ofemulsion does not fade even when it is stored for several months underambient temperature without any protection from light and air. This isdue to the high stability of TPE molecules, which is distinctlydifferent from other dye molecules that are prone to be bleached underroom illumination.

Example 25 Preparation of Fluorescent Polymer Particles

The procedures are just the same as in Example 24, except that the ratioof TPE-COOH decreases to 0.05 wt % relative to the monomer mixture ofmethyl methacrylate, butyl acrylate and 2-hydroxyethyl methacrylate(4:5:1 in volume).

The polymer particle dispersion prepared is quite uniform and stable.The particle dispersion is highly emissive under UV irradiation, even invery dilute state, The photoluminescence spectrum of the dispersion isshown in FIG. 16. The emission peak is found at 458 nm, which issomewhat blue shifted compared to the pure TPE-COOH.

Example 26 Preparation of Fluorescent Polymer Particles

The procedures are just the same as in Example 25, except that the ratioof methyl methacrylate, butyl acrylate and 2-hydroxyethyl methacrylatechanges to 5:4:1 in volume.

The polymer particle dispersion prepared is quite uniform and stable.The particle dispersion is highly emissive under UV irradiation, even invery dilute state. The particle size distribution of the polymernanoparticles in the emulsion was shown in FIG. 18. The diameter of thepolymer nanoparticles is on the average of ˜80 nm, and the sizedistribution is narrow.

Example 27 Preparation of Fluorescent Polymer Particle

The procedures are just the same as in Example 25, except that theemulsifier concentration decreases to 0, that is, the emulsionpolymerization proceeds in the absence of emulsifier.

The polymer particle dispersion prepared is quite uniform. The particlestend to precipitate, however, they are readily be redispersed uponagitation. The particle dispersion is highly emissive under UVirradiation, even in very dilute state. The SEM image of the fluorescentpolymer particles are shown in FIG. 18A, indicating a 760 nm particlesize.

Example 28 Preparation of Fluorescent Polymer Particles

The procedures are just the same as in Example 25, except that theemulsifier concentration decreases to 0.02 wt %.

The polymer particle dispersion prepared is quite uniform and stable.The particle dispersion is highly emissive under UV irradiation, even invery dilute state. The SEM image of the fluorescent polymer particlesare shown in FIG. 18B, indicating a 250 nm particle size.

Example 29 Preparation of Fluorescent Polymer Particles

The procedures are just the same as in Example 25, except that theemulsifier concentration decreases to 0.04 wt %.

The polymer particle dispersion prepared is quite uniform and stable.The particle dispersion is highly emissive under UV irradiation, even invery dilute state. The SEM image of the fluorescent polymer particlesare shown in FIG. 18C, indicating a 120 nm particle size.

Example 30 Preparation of Fluorescent Polymer Particles

The procedures are just the same as in Example 25, except that2-hydroxyethyl methacrylate is replace by acrylic acid.

The polymer particle dispersion prepared is quite uniform and stable.The particle dispersion is highly emissive under UV irradiation, even invery dilute state. The fluorescent particles have carboxyl functionalgroups on the surface, which is favorable to the bioconjugation.

Example 31 Preparation of Fluorescent Polymer Particles

The procedures are just the same as in Example 26, except that2-hydroxyethyl methacrylate is replace by acrylamide.

The polymer particle dispersion prepared is quite uniform and stable.The particle dispersion is highly emissive under UV irradiation, even invery dilute state. The fluorescent particles have amine functionalgroups on the surface, which is favorable to the bioconjugation.

Example 32 Preparation of Fluorescent Polymer Coating

The procedures for preparation of fluorescent dispersion are just thesame as in Example 24. The particle dispersion is highly emissive underUV irradiation, even in very dilute state. The size of the fluorescentparticle is less than 100 nm and the glass transition temperature isbelow room temperature. The dispersion prepared is suitable for filmformation, and the fluorescent coating film is shown in FIG. 19A. Thecoating film formed by control dispersion is nonluminescent while thatformed by the fluorescent particle dispersion is highly emissive underUV irradiation.

Example 33 Preparation of Fluorescent, Free-Standing, Flexible PolymerFilm

The procedures for preparation of fluorescent dispersion are just thesame as that in Example 24. The particle dispersion is highly emissiveunder UV irradiation, even in very dilute state. The size of thefluorescent particle is less than 100 nm and the glass transitiontemperature is below room temperature. The dispersion prepared issuitable for film formation. With a PTFE mold, a free-standing flexiblefilm can be facilely fabricated, and the fluorescent film is shown inFIG. 19B. The coating film fanned by control dispersion isnonluminescent while that formed by the fluorescent particle dispersionis highly emissive under UV irradiation. Such fluorescent free-standingflexible polymer film can be used as flexible organic optoelectronicdevices.

Example 34

The fluorescent polymer nanoparticles with amino groups were preparedwith the method demonstrated in Example 30. The nanoparticle suspensionwas diluted 10 times by minimum essential media, Then 10 mg oftransferrin (Tf) was added into this mixture and gently stirred at roomtemperature for 2 hours to allow the protein to covalently bond to theparticle surface. The human cancer cell lines HeLa was cultured inDulbecco minimum essential media with 10% fetal bovine serum (FBS), 1%penicillin, and 1% amphotericin B. The day before treatment, cells wereseeded in 35 mm culture dishes at a confluency of 70-80%. On thetreatment day, the cells in serum-supplemented media were treated withthe Tf-conjugated nanoparticles for 2 hours at 37 C. Afterwards, thecells were washed three times with PBS and directly imaged using afluorescent microscope.

The results are shown in FIG. 20. From the microscopic fluorescenceimages, it can be seen that the whole cells are bright, indicating thatthe fluorescent nanoparticles have migrated into the cells. In otherwords, they are labeled.

Results on cell labeling using the fluorescent polymer nanoparticleshave been obtained. As shown below, 48 nm fluorescent polymernanoparticles were first prepared with amino groups on the surface, andthen bioconjugation of transferring, a known protein that tends totarget HeLa cells, was carried out. Subsequently, the HeLa cells inserum-supplemented media were treated with the particle-transferrinconjugates. As a result, transferring-conjugated nanoparticles weretransported into the cells through the transferrin receptor mediatedendocytosis pathway. Since transferrin receptors are minimallydistributed in normal cells, transferrin serves as an excellent ligandfor preferentially targeting cancerous cells in vitro and in vivo. Fromthe microscopic fluorescence image (FIG. 20), it can be seen that thewhole cells are bright, indicating that the fluorescent nanoparticleshave migrated into the cells. In other words, they are labeled.

Example 35

In order to utilize the dye molecules as biosensors in aqueoussolutions, we prepared PPS—OH and tested its ability to detect bovineserum albumin (BSA). FIG. 21 depicts the PL spectra of thewater/methanol (6:4) solutions of a PPS—OH in the presence of KOH andBSA at different concentrations. Clearly, with increasing amounts of BSAthe PL intensity increases significantly. Upon prolonged standing the PLintensity enhances further, probably due to the more completeinteraction of PPS—OH with BSA (FIG. 22).

Example 36 Quadruplex Recognition

A complex of ssDNA and TTAPE was prepared by mixing 10 μL G 1 (0.1 mM)and 50 μL TTAPE (0.01 mM) in 5 mM Tris-HCl buffer in a 1.5 ml Eppendorfcup. The solution was incubated at 4° C. for 30 min. G-quadruplexformation was induced by adding 10 μL of a 1.0 M KCl solution into theEppendorf cup. The final concentrations of TTAPE and G1 were kept at 4.5and 9.0 μM, respectively. For other cationic species, the same amountsof corresponding salts were used. Kinetic experiment was conductedimmediately after the injection of the cationic solution, while otherspectral measurements were performed after an incubation period of 30min.

In the aqueous buffer solution, G1 takes a random coil conformation andshows a weak circular dichroism (CD) curve (FIG. 30). Adding K⁺ into theG1 solution (G1/K⁺) promotes G-quadruplex formation, which brings abouta change in the CD spectral profile as well as an increase in theellipticity. The G₁/TTAPE/K⁺ system shows a CD curve with a similarprofile and ellipticity, implying that the dye does not affect thequadruplex conformation. The quadruplex formation induces ˜20 nmred-shift in both excitation and emission spectra of TTAPE (FIG. 31). Aserial titration experiment using K⁺ as titrate reveals that thespectral red-shift starts from [K⁺]˜10 mM and completes at [K⁺]˜100 mM(FIG. 32). The FL intensity at 470 nm, on the other hand, ismonotonically decreased with increasing the K⁺ concentration. Closerinspection of the data finds that the spectrum at high [K⁺] contains ashoulder band at ˜400 nm. This shoulder is probably associated with theemission of the TTAPE molecules that are still bound to the quadruplexbut via only one or two of its four ammonium arms. These partially bounddye molecules may undergo partial intramolecular rotations and thus emitweak light in the blue spectral region.

The addition sequence is systematically investigated to see how itaffects the FL and CD spectra of the TTAPE/quadruplex structure. As canbe seen from the data summarized in Table 2 and FIGS. 33A and 33B, theFL/CD intensities vary in the order of following addition sequence:K⁺->TTAPE/G1>G1->TTAPE/K⁺>TTAPE->G1/K⁺. Comparison of the data inentries 1 and 2 (Table 2) suggests that some TTAPE molecules pre-roundto the G1 strands have been incorporated into the G-quadruplex structureduring the K⁺-induced structural transformation. When TTAPE is addedafter the G-quadruplex has been formed (entry 3), the dye molecules aredifficult to bind to the quadruplex surrounded by numerous K⁺ ions,hence the observed lowest FL and CD intensities. The profiles of the CDspectra for all the G-quadruplexes formed in the three entries arealmost identical (FIG. 33B), confirming that TTAPE does not appreciablydistort the G-quadruplex structure.

TABLE 2 Effect of addition sequence on FL and CD intensities ofTTAPE/quadruplex complexes at room temperature^([a]) Entry Mixture^([b])Additive FL intensity^([c]) CD intensity^([d]) 1^([e]) TTAPE/G1 K⁺ 1.001.00 2 TTAPE/K⁺ G1 0.82 0.92 3 G1/K⁺ TTAPE 0.70 0.88 ^([a])Forcomparison final concentrations of the three components in all themixtures were adjusted to be the same. ^([b])Incubated at 4° C. for 30min. ^([c])Relative intensity at 492 nm. ^([d])Relative intensity at 295nm. ^([e])Data corresponding to those given in FIGS. s 30 and 31.

Example 37

As stated above, addition of K⁺ into TTAPE/G1 results in aquadruplex-specific emission peak at 492 nm. Addition of Na⁺ intoTTAPE/G1, however, quenches this band (FIG. 34A). Similarly, othercationic species including Li⁺, NH₄ ⁺, Mg²⁺ and CA²⁺ all attenuate thisemission band (FIGS. 35A and 35B). After adding into TTAPE/G1, thesecation species competes with TTAPE for binding with G1. The externallyadded cation species prevails because its amount is >10⁴-fold higherthan that of TTAPE. Once the dye molecules are stripped from the DNAstrand, their intramolecular rotations are no longer restricted and theAIE band is thus turned off The FL spectrum of TTAPE/G1 in the presenceof K⁺ is clearly different from, and its peak intensity is outstandinglyhigher than, those in the presence of other cations, suggesting thepotential utility of TTAPE/G1 as a K⁺ biosensor.

Example 38 Effects of DNA Strands

DNA samples of G1, C1 and C2 were obtained from Operon in desalt purityand used without further purification. G1 was chosen as a model ssDNAmimicking the human telomeric repeat sequence d(T₂AG₃), which is capableof forming intramolecular G-quadruplex. Concentrations of the DNAstrands were determined by measuring their absorptivity (E) values at260 nm in a 100 μL quartz cuvette [ε(×10⁵ M⁻¹ cm⁻¹): 2.14 (G1), 1.85(C1), 1.85 (C2)]. Water was purified by a Millipore filtration system.Buffer solution was prepared by titrating 5 mM Tris with HCl until itspH value reached 7.50. All experiments were performed at roomtemperature unless otherwise specified.

If a DNA contains no G unit, its TTAPE complex ceases to show thequadruplex-specific response to K⁺. C1 is also a 21-mer ssDNA, butunlike G1, it possesses no G-rich repeat sequence. When C1 is admixedwith TTAPE, a blue emission at 474 nm is resulted (FIG. 38A). Thisemission is, however, quenched upon addition of other cations includingK⁺. Different from G1, C1 cannot fold into G-quadruplex structure in thepresence of K⁺. The K⁺ ions here just compete with the TTAPE moleculesfor DNA binding, thus resulting in the expulsion of the dye moleculesfrom the C1 strand and the quenching of the light emission.

To be qualified as a specific probe for G-quadruplex recognition, thedye must be able to distinguish the quadruplex from other DNAconformations, especially the double-stranded (ds) one, which is themost ubiquitous conformation for DNAs in living organisms. C1 is acomplimentary strand of G1: the two DNA strands hybridize to form aduplex (FIG. 39). The dsDNA induces TTAPE to emit at 470 nm, which isdifferent from the dye emission in the presence of the G-quadruplex(λ_(em)=492 nm; FIG. 40). The interaction of TTAPE with dsDNA is againelectrostatic in nature: when large amounts of other cations are addedinto G1/C1 solutions, the bound dye molecules are replaced by theexternally added cations and the AIE emission band is accordinglyattenuated (FIG. 41).

For comparison, the quadrulplex/TTAPE complex is mixed with equal molaramounts of its complementary and noncomplementary strands C1 and C2,respectively. The resultant TTAPE/G1/K⁺/Ci mixtures are annealed at ˜58°C., a temperature ˜2° C. below the melting points of the DNAs (˜60° C.)for 15 min, followed by a slow cooling to 25° C. to allow double-helixformation. The hybridization of G1 with C1 unfolds the G-quadruplexstructure and yields a duplex (dsDNA). As a result, thequadruplex-specific emission at 492 nm is quenched (FIG. 40). The duplexis saturated by the prevailingly large amount of K⁺ ions and leaveslittle room for TTAPE molecules to bind, hence making the solutionnon-emissive. Similar results are obtained for other cationic species(FIG. 38B).

Since C2 is noncomplementary to G1, the G-quadruplex remains unperturbedand the emission at 492 nm is preserved. Intriguingly, however, theemission in the bluer region (at ˜400 nm) is increased upon admixingwith C2. Although the whole strand of C2 is non-complementary to G1,partial hybridization of some base units of C2 with those of theG-quadruplex of G1, especially those on its surface, via GC and/or ATbase pairing is possible. Such pairing replaces some, although not all,of the ammonium groups of a TTAPE bound to the G-quadruplex. The dyemolecules hanging on the quadruplex via one or two ammonium arms canundergo partial intramolecular rotations and thus emit in the bluerspectral region. Addition of large amounts of other cationic speciesinto the TTAPE/G1/C2 solutions drives all the dye molecules out of touchwith the DNA strands. As a result, the solutions become non-emissive(FIG. 41).

Example 39 Time-Dependent FL

Time-dependent FL was measured on a FluostarOptima multifunctionalmicroplate reader (BMG Labtechnologies) with λ_(ex)/λ_(em), set at350/470 nm. To gain insight into dynamics of the folding process of G1,time-dependent FL measurements are performed. Solutions containing TTAPEand G1 are first incubated for 30 min to ensure complete dye/DNAcomplexation and transferred to a 96-well microtiter plate afterincubation. Appropriate amounts of metal ions were added by an automaticinjection mode. Kinetic measurements were performed at 20° C. and the FLdata were recorded in every 4 s. The change in the emission intensity(1) of the solution after the addition of K⁺ can be fitted by asecond-order exponential curve:

I=A ₁ e ^(−t/τ1) +A ₂ e ^(−t/τ2) +c  Eq. (1)

where t is the time, τ₁ and τ₂ are the time constants of FL recovery, A¹and A² are the respective amplitudes (the folding process ischaracterized by negative A values), and c is the FL intensity att=infinity. Mean time constant (<τ>) was calculated according to eq. 2:

$\begin{matrix}{{\langle\tau\rangle} = {\frac{{A_{1}\tau_{1}} + {A_{2}\tau_{2}}}{A_{1} + A_{2}}.}} & {{Eq}.\mspace{14mu} (2)}\end{matrix}$

In one embodiment, a solution of potassium chloride is then injected attime t=0 (automatic injection mode) and the emission intensity at 470 nmis monitored. The emission drops abruptly to ˜30% of its originalintensity at the beginning but starts to recover after ˜8 s and finallyreaches a plateau at ˜320 s (FIG. 42). This suggests a very fastion-exchange process between K⁺ and TTAPE with G1 at the beginningBecause of its smaller size and higher concentration, K⁺ outperformsTTAPE in the DNA binding, leading to the initial quick drop in the FLintensity. The G1 strand then starts to fold into quadruplex with theaid of during which the TTAPE molecules are attracted to bind with thequadruplex, as manifested by the recovery of the FL signal after ˜8 s.The complete folding of G1 into the quadruplex conformation takes only˜5 min. This result is consistent with the observations in the previousstudies on the DNA folding processes using the surface plasmon resonanceand electrospray mass spectrometry techniques.

The FL recovery process can be fitted by a double-exponential curve,giving a weighted mean time constant (<τ>) of 116 s. The inverse of <τ>can be viewed as a rate constant, allowing one to have a kinetic pictureof the folding process of the DNA. Control experiments using othercationic species such as Na⁺, NH₄ ⁺ and Ca²⁺ reveal similar FL decreases(FIG. 43), suggesting that the same ion-exchange mechanism is involvedin the dye detachment processes. The emission intensities, however, failto recover from the low values even after a time period as long as 1000s. For Na⁺ and NH₄ ⁺, it is probably due to the geometric unfitness ofTTAPE with the G-quadruplexes formed in the presence of these twocationic species. For Ca²⁺, it is simply because this ion cannot inducethe formation of a G-quadruplex structure.

Example 40 Urinary Protein Detection

Urine is an easily accessible body fluid and contains a complex mixtureof proteins and peptides, which makes it a reliable source of biomarkersfor diagnostics and clinical studies. Determination of urinary proteincomposition is of major clinical importance because it readily reflectsserum composition and kidney functionality. The synthesis route ofTPE-SO3 is described in Example 2 above.

The water-soluble salt TPE-SO3 is expected to be suitable for proteindetection and quantification as an FL bioprobe. A stock solution ofTPE-SO3 (0.5 mM) was prepared by directly dissolving it in a pH 7.0phosphate buffer. The dye solution in the absence of HSA, a modelprotein, is almost non-emissive (FIG. 46A). Its emission is switched oninstantly by the addition of HSA. Its intensity increase (up to˜55-fold, FIG. 46B) and linear range (0-100 ug/ml). In order todetermine whether TPE-SO3 can be used in the medium of urine or not,experiments were performed in artificial urine solution (pH 6.0). Anartificial urine solution was prepared according to the recipe providedby Brooks and Keevil (T. Brooks, C. W. Keevil, Lett. Appl. Microbial.1997, 24, 20 3-997. The artificial urine solution was 1.1 mm lacticacid, 2.0 mm citric acid, 25 mm sodium bicarbonate, 170 mm urea, 2.5 mmcalcium chloride, 90 mm sodium chloride, 2.0 mm magnesium sulfate, 10 mmsodium sulfate, 7.0 mm potassium dihydrogen phosphate, 7.0 mmdipotassium hydrogen phosphate, and 25 mm ammonium chloride all mixed inMillipore water. The pH of the solution was adjusted to 6.0 through theaddition of 1.0 m hydrochloric acid.

The results show that the presence of high concentration of urea andsalts do not interfere with the function of TPE-SO3. The detection limitand linear range can be tuned by adjusting the dye concentration.Intriguingly, TPE-SO3 displays high affinity to albumin over any otherproteins (FIGS. 47A and 47B). Albumin proteins, such as HSA, may have alarge hydrophobic cavity, which may attract the dyes to stay in it,leading to strong binding interaction. On the other hand, other kinds ofprotein, such as trypsin, papain, pepsin, and IgG can only adsorb thedyes on the surface of the protein by electrostatic interaction. Thus,in the medium with high ionic strength may mitigate their interaction,resulting in weak or no FL signals. Cross-contaminant experiments werealso performed (FIG. 48). The results show that the interaction ofTPE-SO3 with HSA can hardly be affected in the presence of otherspecies.

Similar to TTAPE, TPE-SO3 can also be used in PAGE assays for detectingproteins in biological samples. In a prestaining process samples aremixed with TPE-SO3 prior to being loaded into the gel. The results ofthe prestaining process with TPE-SO3 can be seen in top left portion ofFIG. 25. In addition, a poststaining process was performed where afterelectrophoresis, the gel was immersed into TPE-SO3 solution for 5 minsbefore taking an image. The results of the post-staining process withTPE-SO3 can be seen in the top right portion of FIG. 25.

Coomassie Brilliant Blue is a blue dye that can bind to the proteins ofa within the PAGE assay and allow you to directly visualize them. AfterCoomassie staining and destaining, the proteins will appear as bluebands as shown in the lower right portion of FIG. 25. CoomassieBrilliant Blue staining requires long reaction time (normally soakedmore than 6 hours), destaining step (immersed in acid solution todissolve the unbound dyes), and low sensitivity (colorimetric-basedmethod) (see U.S. Pat. No. 5,922,186). In contrast, TPE-SO3 offers aneasy and sensitive way to do this same job. Gels only need to be exposedto TPE-SO3 for 3-5 minutes to make the protein bands visible. The use ofTPE-SO3 requires no destaining step. The sensitivity of TPE-SO3 is muchhigher than that of Coomassie Brilliant Blue. It is clear that the useof TPE-SO3 as a stain for PAGE assays is a faster method with fewersteps that provides greater sensitivity than conventional stainingmethods for protein detection.

Example 41 TTAPE Also Acts as a G-Quadruplex Inducer

CD spectral profiles of 9 μM DNA in 5 mM Tris-HCl buffer solutions (pH7.5) incubated under 25° C. with four different TPE derivatives at 4.5μM or with 0.5M K⁺ ions are studied (FIG. 77). TPE derivatives 1-4 areTTAPE (Example 6), TPE-2 (chemical structure is not shown herein), TPE-3(chemical structure is not shown herein) and TPE-C2N+(Example 3),respectively. Results reveal that TPE-2, TPE-3 and TPE-4 result inspectral profile comparable to the negative control, where DNA incubatedwith neither TPE derivatives nor K⁺ ions. On the other hand, DNAincubated with TTAPE or the known quadruplex inducer; K⁺ ions have verysimilar spectral profiles, while the ellipticities of DNA/TTAPE aresomehow weaker than DNA/K⁺. This study leads to the surprisinglyfounding that TTAPE acts as an internal quadruplex inducer under 25° C.,making it useful as a quadruplex-targeting drug for anti-cancer therapy.

Example 42 TPE-SO3 is Highly Sensitive and Specific to Amyloid Fibrils

Bovine insulin powder was dissolved in 25 mM NaCl/HCl solution (pH 1.6at 65° C.). The solution was passed through a 0.45 μm filter and theconcentration was determined by measuring its absorbance at 278 nm.Stock solution of TPE-SO3 with a concentration of 1.0 mM was prepared bydissolving appropriate amount of dye in PBS solution (pH 7.0). Insulinfibrils were prepared by incubating the protein (320 μM) in buffersolution (pH 1.6) at 65° C. for 20 h.

The TPE-SO3 solution alone in phosphate buffered saline (PBS) where noprotein is present is non-emissive. The solution remains faintlyluminescent at ca. 470 nm upon addition of native bovine insulin. TheTPE-SO3 solution becomes luminescent in the presence of small amount offibrillar insulin (FIG. 66A). The emission intensity is proportional toconcentration of fibrillar insulin as seen by the progressivelyenhancement of intensity with an increase in the concentration offibrillar insulin. The rate of fluorescence enhancement (I/I_(o)) isfast at low fibrillar insulin concentration and becomes nearly constantat fibrillar insulin concentration greater than 20 μM. The fluorescenceof TPE-SO3 remains negligible even when the concentration of nativeinsulin is up to 100 μM (FIG. 66B). To further evaluate the specificityof TPE-SO3 towards fibrillar insulin, the rate of FL intensityenhancement of TPE-SO3 in insulin mixture with different molar fractionsof fibrillar insulin (ti) are measured where the total proteinconcentration is kept at 5 μM. Results (FIG. 66B inset) shows thatemission of TPE-SO3 increases monotonically with an increase in thefibrillar insulin fraction in a linear manner, indicating that thefluorescence of TPE-SO3 in detecting insulin fibrils is independent onthe presence of native insulin.

The distinct native insulin independent fluorescence emissioncharacteristic of TPE-SO3 to fibrillar insulin enables the compound tobe used as an indicator for monitoring the kinetics of insulinfibrillation. As shown in FIG. 68A, insulin solution dissolved inHCl/NaCl (pH 1.6) is incubated at 65° C., such elevated temperature andlow pH conditions favor the growth of amyloid fibrils of insulin. FLmeasurement is carried out at regular time intervals up to 60 mins,where an aliquot of insulin is taken out and diluted with PBS (pH 7.0)at room temperature and an aliquot of TPE-SO3 is added to the proteinsolution before spectral measurement. All FL measurements are conductedin a PBS buffer (pH=7.0); [TPE-SO3]=15 μM; [Insulin]=5 μM and λ_(ex)=350nm.

Results shows that no FL signal is recorded when insulin has beenincubated for less than 20 mins. The solution becomes emissive at anincubation time from 30 mins and emission increases rapidly and reachesmaximum at 60 mins incubation time. The change in fluorescence intensityat different incubation times corresponds to insulin fibrillation. Thechanges in FL intensity recorded at 470 nm at different time intervalsreveals the duration of (I) lag phase, (II) growth phase and (III)equilibrium phase during fibrillation can be seen in FIG. 68B.

Example 43 TPE-SO3 is an In-Situ Inhibitor of Insulin Fibrillation

In the study of TPE-SO3 perturbation on insulin fibril formation,different concentrations of TPE-SO3 are admixed with insulin solution inbuffer solution (pH 1.6) prior to incubation at 65° C. and thefluorescence of TPE-SO3 during the time course of fibrillation isdetermined. It is surprisingly shown that prior incubation of TPE-SO3influences the kinetics of insulin fibril formation (FIG. 69A). TPE-SO3lengthens the lag phase and decelerates the elongation rate of growthphase during fibrillation. The lag phase which associates with aconstant low FL signal is extended from 30 mins to 2 h when insulin ispremixed with 10 μM TPE-SO3. The exponential growth phase is alsoprolonged from 30 to 90 mins. It takes more than 3 h in order to reachto the equilibrium phase, while it takes only 1 h in the absence ofTPE-SO3.

The retardation effect of each of TPE-SO3 and BSPOTPE on amyloid fibrilformation strengthens with a higher concentration of TPE-SO3. FIG. 69Bshows the correlation between the lag time defined as the duration oflag phase, and the TPE-SO3 concentration incubated with insulinsolution. The lag time is shown to extend linearly with an increase inTPE-SO3 concentration. At 25 and 50 μM TPE-SO3, the lag time is extendedto 3 and 6 h, respectively. Further increment of TPE-SO3 concentration(100 μM) suppress the formation of fibrils, as revealed by noenhancement in FL signal even after incubation for 20 h. The retardationeffect of TPE-SO3 is further confirmed by the exponential decrease ofinsulin fibrillation growth rate as the concentration of TPE-SO3 becomeshigher (FIG. 69C). TPE-SO3 behaves as an internal inhibitor of amyloidfibrillation is clearly seen. Similar effects are seen for BSPOTPE inFIGS. 81A and 81B.

Images from scanning electron microscopy (SEM) and transmission electronmicroscopy (TEM) further confirm the above observation. In the absenceof TPE-SO3, amyloid fibrils with diameters of ˜20 nm are observed insolution after 1 h incubation (FIGS. 74A and 74B). No fibril-likestructure is, however, found under the same condition in the presence ofTPE-SO3 (FIG. 74C), indicating that the fibrillation is still in the lagphase after 1 h. After 7 h incubation, mature amyloid fibrils areobserved in incubated insulin solutions with and without TPE-SO3 (FIG.74D-F).

Example 44 TPE-SO3 does not Affect Fibrillation Once Fibril Starts toGrow

TPE-SO3 is added at different time points of incubation under fibrilformation favorable condition at pH 1.6 and 65° C. TPE-SO3 is added tothe insulin solution to afford a final concentration of 10 μM. FIG. 73shows the resultant time-course curve where TPE-SO3 is added atdifferent time intervals as compared to the fibrillation in the absenceof TPE-SO3. Addition of TPE-SO3 after 10 mins incubation induces asignificant retardation effect on the insulin fibrillation (FIG. 75A).When TPE-SO3 is added at 20 mins, only slight retardation effect can beobserved (FIG. 75B). No effect is observed at all when the dye moleculeis added after 30 mins of incubation (FIG. 75C). The results indicatethat the lag phase where the fibril-competent nucleation occurs is therate-determining step for the insulin fibrillation. On condition thatTPE-SO3 is added before the formation of fibril-competent nucleus, thefibrillation can be inhibited as reflected by extension of the lagphase. At short incubation time such as 10 min, partially unfoldedinsulin monomers are formed in the solution. The presence of TPE-SO3will interact with these unfolding intermediates and slow down furthernucleation evolvement and subsequent oligomer formation. At a longerincubation time of 30 min, critical nuclei are formed from partiallyunfolded intermediates. The elongation is so favorable that the additionof TPE-SO3 has no impact on the kinetic of the fibril formation. Oncethe fibrils start to grow, the presence of TPE-SO3 will not terminatefibrillation or dissociate the fibrils.

Example 45 Emission of TPE-SO3 is pH Dependent

To understand the effect of pH in TPE-SO3 fluorescence, the FL emissionsof TPE-SO3 to native and fibrillar forms of insulin at different pHconditions are determined (FIG. 76). The emission of TPE-SO3 withinsulin increases gradually as the pH of surrounding condition lowers,indicating that electrostatic attraction is an indispensable drivingforce for the binding of TPE-SO3 to insulin. Owning to the low pKa ofsulfonate groups, TPE-SO3 has a net negative charge under a wide rangeof pH conditions. The isoelectric point (pI) of bovine insulin is pH5.6; therefore at pH lower than pH 5.6, insulin carries a net positivecharge. Accordingly, the negatively charged TPE-SO3 can bind stronglywith insulin via electrostatic attraction, leading to the strongfluorescence emission.

Example 46 Amyloid Fibril Imaging Using TPE-SO3

Amyloid fibrils stained with TPE-SO3 solution can be visualized underfluorescence microscope. Microaggregates of insulin fibrils are easilyidentified as bright greenish-blue emission from TPE-SO3 (FIG. 67).Owing to the excellent water solubility of TPE-SO3, fluorescencebackground can be ignored and fibril-like structures can be discerned athigh magnification (FIG. 67 inset). These results confirm theutilization of TPE-SO3 as a FL stain for protein aggregates from tissuesections ex vivo. Similar results are seen for Cy₂Silo (FIG. 82), TPEBe(FIG. 84), and TPE-MitoY (FIG. 85), indicating that these can all beutilized as FL stains for protein aggregates from tissue sections exvivo as well.

Example 47 TPE-SO3 is Highly Specific to Fibrillar Amyloid Proteins

To evaluate the specificity of TPE-SO3 fluorescence characteristic tofibrillar proteins, fluorescence intensities of TPE-SO3 against otherprotein monomers are tested. A stock solution of TPE-SO3 with aconcentration of 50/1 is mixed with different protein monomer solutionsat 1.0 mg/mL. The fluorescence intensities of the differentTPE-SO3-protein mixtures are measured. PBS buffer at pH 7.0 and solutionof TPE-SO3 are used as negative controls. Results (FIG. 77) shows thatFL of TPE-SO3 with heparin, apoferritin and cellulose is comparable toTPE-SO3 solution alone; FL with papain is comparable to native insulinmonomer and fibrillar insulin is significantly higher than othercellular proteins tested.

Not only does TPE-SO3 fluoresce against fibrillar insulin, but otherfibrillar amyloid proteins. To illustrate, the emission of TPE-SO3 at500/1 is measured at λ_(ex)=350 nm with 0.1 mg/mL amyloid-β-peptidebefore and after aging. It is shown that the emission of TPE-SO3increases dramatically upon the formation of amyloid-β-peptide fibrils(i.e., after aging) (FIG. 72). Moreover, the rate of fluorescenceenhancement of TPE-SO3 (10 μM, 50 μM and 100 μM) at different timepoints during lysozyme fibril formation over 4 days (two collectionseach day) are determined (FIG. 73). As seen in FIG. 73, TPE-SO3 producesfluorescence profile corresponding to lysozyme fibrillation verifyingits usefulness as an external indicator for monitoring amyloid proteinfibrillation.

Example 48 TPE-SO3 is Non-Toxic and Stable

The toxicity of TPE-SO3 is evaluated using methyl thiazolyl tetrazolium(MTT) assay. 5×10³ cells per 0.1 ml per well of HeLa cells are treatedwith different concentrations of TPE-SO3 (5, 10, 20, 40, and 80 μM) in a96-well plate for 48 hr at 37° C. 20 ml of PBS containing3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; 5mgmL⁻¹) is added to each well and subject to further incubation at 37°C. for 2-4 h. To dissolve intracellular formazan produced by activemitochondria in HeLa cells, detergent solution (10% w/v sodium dodecylsulfate in 10 mm HCl; 100 mL per well) is added to each well andincubated overnight in the dark at room temperature. To determine therelative cell viability, absorbance at 595 nm is measured using aspectrophotometric plate reader. It is demonstrated that the toxicity ofTPE-SO3 is negligible (FIG. 70).

Furthermore, it is observed that the TPE-SO3 compound is stable underambient condition for more than one year and aqueous solution thereofremain unchanged at 4° C. refrigeration for more than six months. Inaddition to TPE-SO3's internal retardation effect on amyloid proteinmonomer to aggregate, the stability and non-toxicity characteristicsthereof confirm the usefulness of compound TPE-SO3 to be used asantiamyloid agent in storing and delivery therapeutic amyloids, such asinsulin.

Based on the above examples, while amyloid protein is used as anexample, it is to be understood that the external AIE effect andinternal retardation effect of the subject water-soluble conjugatedpolyene compounds are also applicable to other protein aggregates.

With the information contained herein, various departures from precisedescriptions of the present subject matter will be readily apparent tothose skilled in the art to which the present subject matter pertains,without departing from the spirit and the scope of the below claims. Thepresent subject matter is not considered limited in scope to theprocedures, properties, or components defined, since the preferredembodiments and other descriptions are intended only to be illustrativeof particular aspects of the presently provided subject matter. Indeed,various modifications of the described modes for carrying out thepresent subject matter, which are obvious to those skilled in chemistry,biochemistry, or related fields are intended to be within the scope ofthe following claims.

We claim:
 1. A method of monitoring amyloid protein fibrillation in abiological sample comprising contacting a water-soluble conjugatedpolyene compound that exhibits aggregation induced emission with thebiological sample containing an amyloid protein; and detectingfluorescence emitted by said water-soluble conjugated polyene compoundand the fluorescence emitted is induced by aggregation of saidwater-soluble conjugated polyene compound when contacting with saidbiological sample, wherein said water-soluble conjugated polyenecompound is selected from the group consisting of:1,2-Bis[4-(3-sulfonatopropoxy)phenyl]-1,2-diphenylethene sodium;1,2-Bis[4-(3-triphenylphosphonium)phenyl]-1,2-diphenylethene bromide;1-[4-(1-methyl-4-pyridine)ethene]-1,2,3-triphenylethenehexafluorophosphate;1-[4-(1,2-dimethyl-5-benzothiazole)]-1,2,3-triphenylethene iodide;N,N′,N″,N′″-[1,2-tetrakis(1,4-phenoxyethyl)vinyl]tetrakis(trimethylammoniumbromide);1-[4-(1-ethyl-2-benzothiazole)ethene]-1,2,3-triphenylethenehexafluorophosphate; and1,1′-methyl-2,5-bis[4-(1-sulfonatopropyl-2-isoindole)ethenephenyl]-3,4-bisphenylsilole.
 2. The method of claim 1, wherein said amyloid protein isselected from the group consisting of insulin, amyloid betapeptide, tau,alpha-Synuclein, PrP and polyglutamine-containing protein.
 3. The methodof claim 1, wherein said contacting is carried out at a pH value equalto or lower than 5.6.
 4. The method of claim 1, wherein said detectingcomprises measuring fluorescence intensity at 470 nm.
 5. The method ofclaim 1, wherein said biological sample is selected from the groupconsisting of a tissue sample, a cell sample, blood, saliva, spinalfluid, lymph fluid, vaginal fluid, seminal fluid and urine.
 6. Themethod of claim 1, wherein said cation is selected from the groupconsisting of K⁺, Li⁺, Na⁺, Mg²⁺, NH₄ ⁺ and Ca²⁺.
 7. A method ofretarding fibrillation of an amyloid protein comprising contacting saidamyloid protein with the water-soluble conjugated polyene compound ofclaim 1 to form a stabilized amyloid protein.
 8. The method of claim 7,further comprising converting said amyloid protein from native form intomonomer thereof in an acidic environment prior to said contacting. 9.The method of claim 7, further comprising increasing concentration ofsaid water-soluble conjugated polyene compound to form a partiallyunfolded amyloid protein associated with said water-soluble conjugatedpolyene compound.
 10. The method of claim 7, wherein said amyloidprotein is selected from the group consisting of insulin, amyloidbetapeptide, tau, alpha-Synuclein, PrP and polyglutamine-containingprotein.
 11. The method of claim 7, wherein said cation is selected fromthe group consisting of K⁺, Li⁺, Na⁺, Mg²⁺, NH₄ ⁺ and Ca²⁺.
 12. Themethod of claim 7, wherein said incubating is carried out at a pH valueequal to or lower than 5.6.
 13. The method of claim 7, wherein saidamyloid protein is a pharmaceutical insulin being administered to asubject.
 14. The method of claim 13, wherein the subject is in need oftreatment for diabetes.
 15. A method of storing and delivering anamyloid protein containing pharmaceutical composition for treatment ofdiseases comprising the method of claim 7.